Human voltage gated sodium channel beta1A subunit and methods of use

ABSTRACT

DNAs encoding human voltage gated sodium channel β1A subunit have been cloned and characterized. The recombinant protein is capable of forming biologically active protein. The cDNA&#39;s have been expressed in recombinant host cells that produce active recombinant protein. The recombinant protein is also purified from the recombinant host cells. In addition, the recombinant host cells are utilized to establish a method for identifying modulators of the receptor activity, and receptor modulators are identified.

This case claims priority to U.S. Provisional Patent Application No.60/294,405 filed Jun. 7, 2000 and entitled “DNA Encoding Human VoltageGated Sodium Channel β1A Subunit” and U.S. Provisional PatentApplication No. 60/236,664 filed Sep. 29, 2000 and entitled “DNAEncoding Human Voltage Gated Sodium Channel β1A Subunit.”

BACKGROUND OF THE INVENTION

Rapid entry of sodium ion into cells causes depolarization andgeneration of the action potential. Such entry of sodium ions inresponse to voltage change on the plasma membrane in excitable cells ismediated by voltage gated sodium channels (VGSC). Therefore, voltagegated sodium channels play a fundamental role in the control of neuronalexcitability in the central and peripheral nervous systems. The VGSC isa protein complex comprising at least a large (200-300 kDa), poreforming, α subunit and two small (30-40 kD) regulatory β1 and β2subunits (Catterall, 1992 & 1993; Isom et al., 1992; Isom et al., 1995).It is well known that VGSC α subunits determine the basic properties ofthe channel because expression of the α subunit of VGSC alone in theheterologous expression systems such as HEK cells, CHL cells and Xenopusoocytes is sufficient to synthesize a functional, but altered, sodiumchannel. Co-expression of VGSC β1 and β2 subunits with the α subunitwill usually normalize the channel properties in heterologous expressionsystems. In order words, VGSC β1 and β2 subunits modulate almost all theaspects of the channel properties including voltage dependent gating,activation and inactivation, and increase the number of functionalchannels on the plasma membrane. The VGSC β subunits may also be therate limiting step controlling the increased expression of sodiumchannels (Caterall, 1992; Isom et al., 1994)

Molecular cloning studies have demonstrated that there are manydifferent types of VGSC α subunits, which can be categorized based ontheir sensitivity to neurotoxin and tetrodotoxin (TAX) (Marban, et al.1998). Because brain VGSC type I, IIA, III, skeletal muscle type I,sodium channel protein 6 (SCP6), its closely homologous peripheral nerve4 (PN4), and peripheral nerve 1 (PN1) are blocked by TTX at nanomolarconcentrations, they are termed TTX-sensitive (TTX-S) sodium channels.The cardiac sodium channel (H1), and peripheral nerve 3 (PN3/SNS) andNaN/SNS2 are normally blocked by TTX in the micromolar range, and aretermed TTX-resistant (TTX-R) sodium channels.

The studies of VGSC β subunits are far behind those of VGSC α subunits.So far, only two types of β subunits, β1 and β2 (Isom, et al. 1992, 1994& 1995) have been cloned and characterized. Recently, a novel VGSC β1Asubunit, a splicing variant of the β1 subunit, has been identified fromrat (Kazen-Gillespie, et al. 2000). Rat VGSC β1A subunit results from anapparent intron retention event. Analysis of rat genomic DNA indicatedthe divergent region (carboxyl region) of β1A is encoded by intron 3with an in-frame termination codon. Like the VGSC β1 subunit, the β1Asubunit increases sodium current density and [³H]Saxitoxin bindingsites, and modulates voltage dependent activation and inactivation ofthe type IIA of VGSC. More interestingly, the expression level andpattern of the VGSC β1A in dorsal root ganglia (DRG) are changedsignificantly in the Chung animal neuropathic pain model (see below),which is consistent with the observation that the sodium current isincreased after nerve injury. Both VGSC β1 and β1A subunits are integralmembrane glycoproteins (Isom, et al. 1992, Kazen-Gillespie, et al. 2000)containing a single transmembrane domain at the carboxyl terminus and anextracellular amino-terminal immunoglobulin-like fold motif maintainedby a single putative disulfide bridge between two highly conservedcysteine residues. VGSC β1 and β1A can be classified as members of theV-set of the Ig superfamily, which includes many cell adhesion molecule,suggesting that β1 and β1A subunits play roles not only in modulatingsodium channel properties, but also in protein targeting and celladhesion (Isom and Catteral, 1996).

An increase in the rate of spontaneous firing in neurons is oftenobserved in peripheral sensory ganglia following nerve injury (Ochoa andTorebjork, 1980; Nordin et al., 1984; Devor, 1994; Woolf, 1994). It hasbeen suggested that this hyperexcitability in neurons is due to alteredsodium channel expression in some chronic pain syndromes (Tanaka et al.,1998). Increased numbers of sodium channels leading to inappropriate,repetitive firing of the neurons have been reported in the tips ofinjured axons in various peripheral nervous tissues such as the DRGwhich relay signals from the peripheral receptors into the centralnervous system (Waxman and Brill, 1978; Devor et al., 1989; Matzner andDevor, 1992; Devor et al., 1992; England et al., 1994; Matzner andDevor, 1994; England et al., 1996). Transcripts encoding the α-IIIsubunit, which are present at only very low levels in control DRGneurons, are expressed at moderate to high levels in axotomized DRGneurons together with elevated levels of α-I and α-II mRNAs (Waxman etal, 1994). Conversely, transcripts of sodium channel α-SNS aredown-regulated in DRG neurons following axotomy (Dib-Hajj et al., 1996).Furthermore, the partial efficacy of sodium blocking agents is welldocumented in patients treated for neuropathic pain (Chabel et al.,1989; Devor et al., 1992; Omana-Zapata et al., 1997; Rizzo, 1997),providing an important link between increased sodium channel expressionand neuropathic pain. Therefore, alterations in sodium channelexpression and subsequent function may be a key molecular eventunderlying the pathophysiology of pain after peripheral nerve injury.

Recently the VGSC β1A subunit had been cloned and was reported toincrease sodium current density at the plasma membrane when co-expressedwith αIIA subunits in CHL fibroblasts (Kazan-Gillespie et al., 2000).β1A is developmentally regulated in the brain, but its potential role inneuropathic pain has not been previously explored. Therefore, theexpression of β1A protein in DRG neurons using the Chung model ofneuropathic pain (Kim and Chung, 1992) was investigated using apolyclonal antibody directed against a unique extracellular region ofβ1A not present in β1. Immunohistochemistry and computer-assisted imageanalysis documented significant up-regulation of VGSC β1A and β1subunits following neuronal injury, compared to very low levels in theDRG from sham operated animal. The distinct punctate and membranelabeling distribution of β1A following peripheral nerve injury suggestedactive translation and possible accumulation into the plasma membrane,unlike β1, where the subcellular distribution remained diffuse.

To further explore the functions of VGSC β1A subunit, human VGSC β1Asubunit has been cloned and characterized in this invention.

SUMMARY OF THE INVENTION

A DNA molecule encoding human VGSC β1A subunit has been cloned andcharacterized, and it represents a novel isoform of the human VGSC β1Asubunit that is preferentially expressed in tissues important forneurological function. Using a recombinant expression system, functionalDNA molecules encoding the channel subunit have been isolated. Thebiological and structural properties of these proteins are disclosed, asis the amino acid and nucleotide sequence. The recombinant DNA moleculesand portions thereof, are useful for isolating homologues of the DNAmolecules, identifying and isolating genomic equivalents of the DNAmolecules, and identifying, detecting or isolating mutant forms of theDNA molecules.

Further, the β1A subunit is implicated in neuropathic pain as evidencedusing an established neuropathic pain model.

In a first aspect of the invention, the invention relates to an isolatednucleic acid molecule that encodes a human β1A sodium channel subunitprotein, said polynucleotide comprising a member selected from a groupconsisting of: (a) a polynucleotide having at least a 75% identity to apolynucleotide encoding a polypeptide consisting of amino acids 1 to 268of SEQ.ID.NO.:14; (b) a polynucleotide having at least 75% identity to apolynucleotide encoding a polypeptide consisting of amino acids 150 to268 of SEQ.ID.NO.:14; (c) a polynucleotide which is complementary to thepolynucleotide of (a) or (b); and (d) a polynucleotide comprising atleast 15 sequential bases of the polynucleotide of (a), (b), or (c). Inone embodiment, the polynucleotide is RNA and in another embodiment, thepolynucleotide is DNA. In another embodiment, the nucleotide sequence isselected from a group consisting of: (SEQ.ID.NO.:12) and(SEQ.ID.NO.:13). A further group includes isolated DNA moleculesconsisting of allelic variants, mutants, and functional derivatives of(SEQ.ID.NO.:12) and (SEQ.ID.NO.:13). In another embodiment, the isolatedDNA molecule is genomic DNA.

The invention further relates to an expression vector for expression ofa human β1A sodium channel subunit protein in a recombinant host,wherein said vector contains a recombinant gene encoding a human β1Asodium channel subunit protein and functional derivatives thereof. Inone embodiment, the expression vector contains a cloned gene encoding aHuman β1A sodium channel subunit protein, having a nucleotide sequenceselected from a group consisting of: (SEQ.ID.NO.:12) and (SEQ.ID.NO.:13)and in another embodiment, the group further consists of allelicvariants, mutants, and functional derivatives of SEQ.ID.NO.:12 andSEQ.ID.NO.:13. In another embodiment, the expression vector containsgenomic DNA encoding a Human β1A sodium channel subunit protein.

The invention also relates to a recombinant host cell containing arecombinantly cloned gene encoding Human β1A sodium channel subunitprotein or a functional derivative thereof. Preferably the gene has anucleotide sequence selected from a group consisting of:(SEQ.ID.NO.:12); (SEQ.ID.NO.:13); and functional derivatives thereof. Inanother embodiment, the cloned gene is genomic DNA.

The invention also contemplates isolated protein encoded by a nucleicacid sequence capable of hybridizing under stringent hybridizationconditions to a nucleotide sequence having the sequence of SEQ ID NO:12or SEQ ID NO:13 that when combined with a Human a sodium channel subunitprotein in a cell permits sodium ion flux in the cell. In oneembodiment, the protein has an amino acid sequence selected from a groupconsisting of: (SEQ.ID.NO.:14) and functional derivatives thereof. Theinvention also contemplates antibodies that are specific to theseproteins and monospecific antibodies, that is, antibodies that will onlyidentify the human proteins of this invention, such as those antibodiesthat specifically recognize a human β1A sodium channel subunit protein.

The invention relates to a process for expression of a Human β1A sodiumchannel subunit protein in a recombinant host cell, comprising the stepsof; (a) introducing an expression vector comprising a nucleic acidsequence capable of hybridizing under stringent hybridization conditionsto a nucleotide sequence, or its complementary sequence, having thesequence of SEQ ID NO:12 or SEQ ID NO:13 into a cell; (b) culturing thecell of step (a) under conditions which allow expression of a proteinencoded by the nucleotide sequence.

The invention further relates to a method of screening for a modulatorof sodium channel activity comprising: (a) providing a cell thatco-expresses a protein encoded by a nucleic acid capable of hybridizingunder stringent hybridization conditions to a nucleotide sequence, orits complementary sequence, represented by SEQ ID NO:12 or SEQ ID NO:13and a sodium channel α subunit protein wherein the cell elicits a sodiumion flux; (b) contacting the cell with a putative β1A modulatingcompound; and (c) measuring a change upon the cell that alters thesodium ion flux. In one embodiment of this method, at least one of theproteins is a recombinant protein. The the change in sodium ion flux ispreferably selected from a group consisting of: (a) increasing thecapacity to open the Na channel; (b) decreasing the capacity to open theNa channel; (c) increasing the rate of desensitization; (d) decreasingthe rate of desensitization; (e) increasing the rate of re-sensitizationof the channel; (f) decreasing the rate of re-sensitization of thechannel; (g) increasing the level of β1A protein expression; (h)decreasing the level of β1A protein expression; (i) increasing the levelof the α/β1A complex protein expression; and (j) decreasing the level ofthe α/β1A complex protein expression. Compounds identified by thesemethods are also contemplated within the scope of this invention as arepharmaceutical composition comprising these compounds.

The invention further relates to a number of methods for treatinganimals, including a method of treating neuropathic pain in a patient inneed of such treatment comprising administration a compound of thisinvention. Methods for treating neuropathic pain in a patient in need ofsuch treatment comprising altering the level of a human β1A subunit in adorsal root ganglia cell in the patient are also contemplated in thisinvention.

The invention also relates to a method of treating chronic pain in apatient in need of such treatment comprising administering a compound ofthis invention as well as a method of treating febrile seizures in apatient in need of such treatment comprising administering a compound ofthis invention.

Methods for treating general epilepsy by administering a compound ofthis invention are contemplated as are anticonvulsant pharmaceuticalcomposition comprising a compound of this invention. Further methodsinclude a method of treating arrhythmia in a patient in need of suchtreatment comprising administering a compound identified by a method ofthis invention and methods for providing local anesthesia to a patientby administering a pharmaceutical composition comprising a compound ofthis invention. The invention further relates to a method for decreasingneuropathic pain in an individual comprising administering to saidindividual a modulator of a sodium channel β1A subunit in an amounteffective to change the sodium channel activity in said individual andto methods for decreasing the expression of sodium channel β1A subunitin the cells of the individual. The invention also relates to a methodfor treating neuropathic pain in a subject comprising altering the levelof sodium channel β1A subunits on the surface of a cell in the subject.

-   -   30. A method for decreasing neuropathic pain in a human        comprising the step of administering a sodium channel β1A        subunit-binding molecule to a sodium channel β1A        subunit-expressing cell in the human.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1—The amino acid sequence of human voltage gated sodium channel β1Asubunit aligned with (A) the rat voltage gated sodium channel β1Asubunit and (B) human voltage gated sodium channel β1 subunit are shown.The overall identity between human and rat VGSC β1A subunits is about72%, while the identity of their carboxyl terminal regions is less than33%. The overall identity of human VGSC β1A and β1 subunits is 75%, butthe identity of their carboxyl terminal regions is less than 17%.

FIG. 2 illustrates the genomic and transcriptional organization of thehuman β1 gene (SCN1B). The gene encoding sodium channel β1 and β1Asubunits spans about 9-10 kb on chromosome 19, containing seven exons(1, 2, 3, 4A, 4, 5, and 6) and five introns. An alternative exon 4A islocated in the 5′ region of the intron 3. The human β1 subunit isencoded by exon 1-3 and 4-6, while human β1A by exon 1-3 and exon 4A.

FIG. 3 demonstrates co-expression of the β1A and the Na_(V)1.2 subunitsin Xenopus oocyte. Peak ionic current conducted by Na_(V)1.2 wasdose-dependently increased by its coexpression with the β1A subunit inoocytes. The cRNAs ratio of Na_(V)1.2 and the β1A subunits are indicatedat the bottom of the bars. The data were averaged from the number ofoocytes shown on the top of the bars. The error bars represent the SEM.

FIG. 4 illustrates the intensity of β1A labeling in DRG neurons innormal rats and in SNL rats at various times post-surgery. The barsshown represent labeling in nociceptive (open bars) and sensory (shadedbars) neurons. N=3-5 animals per group and 30-40 neurons of each typeper animal. The intensity of the labeling in each neuronal type at eachDRG level increased with time post surgery (see table 1 for statisticalanalysis).

FIG. 5 illustrates the intensity of β1 labeling in DRG neurons in normalrats and in SNL rats at various times post-surgery. The bars shownrepresent labeling in nociceptive (open bars) and sensory (shaded bars)neurons. N=3-5 animals per group and 30-40 neurons of each type peranimal. The intensity of the labeling varied with the time post surgery(see table 1 for statistical results).

DETAILED DESCRIPTION

Definitions

The term “protein domain” as used herein refers to a region of a proteinthat can fold into a stable three-dimensional structure independent ofthe rest of the protein. This structure may maintain a specific functionwithin the protein including the site of enzymatic activity, creation ofa recognition motif for another molecule, or provide necessarystructural components for a protein to exist in a particularenvironment. Protein domains are usually evolutionarily conservedregions of proteins, both within a protein family and within proteinsuperfamilies that perform similar functions. The term “proteinsuperfamily” as used herein refers to proteins whose evolutionaryrelationship may not be entirely established or may be distant byaccepted phylogenetic standards, but shows similar three dimensionalstructure or displays unique consensus of critical amino acids. The term“protein family” as used herein refers to proteins whose evolutionaryrelationship has been established by accepted phylogenic standards.

The term “fusion protein” as used herein refers to protein constructsthat are the result of combining multiple protein domains or linkerregions. Fusion proteins are most often created by molecular cloning ofthe nucleotide sequences to result in the creation of a newpolynucleotide sequence that codes for the desired protein.Alternatively, creation of a fusion protein may be accomplished bychemically joining two proteins together.

The term “linker region” or “linker domain” or similar such descriptiveterms as used herein refers to stretches of polynucleotide orpolypeptide sequence that are used in the construction of a cloningvector or fusion protein. Functions of a linker region can includeintroduction of cloning sites into the nucleotide sequence, introductionof a flexible component or space-creating region between two proteindomains, or creation of an affinity tag for specific moleculeinteraction. A linker region may be introduced into a fusion proteinwithout a specific purpose, but results from choices made duringcloning.

The term “cloning site” or “polycloning site” as used herein refers to aregion of the nucleotide sequence contained within a cloning vector orengineered within a fusion protein that has one or more availablerestriction endonuclease consensus sequences. These nucleotide sequencescan be introduced into other cloning vectors to facilitate cloning, thecreation of novel fusion proteins, or they can be used to introducespecific site-directed mutations. It is well known by those in the artthat cloning sites can be engineered at a desired location by silentmutation, conserved mutation, or introduction of a linker region thatcontains desired restriction enzyme consensus sequences. It is also wellknown by those in the art that the precise location of a cloning sitecan be flexible so long as the desired function of the protein orfragment thereof being cloned is maintained.

The term “tag” as used herein refers to a nucleotide sequence thatencodes an amino acid sequence that facilitates isolation, purificationor detection of a fusion protein containing the tag. A wide variety ofsuch tags are known to those skilled in the art, and are suitable foruse in the present invention. Suitable tags include, but are not limitedto, HA peptide, polyhistidine peptides, biotin/avidin, and otherantibody epitope binding sites.

Isolation of Human Voltage Gated Sodium Channel β1A Subunit Nucleic Acid

The voltage gated sodium channel is a multi-subunit protein complexcontaining a pore forming subunit α, and two regulatory subunits, β1 andβ2. While the α subunit determines the basic properties of the channel,β1 and β2 subunits modulate almost all aspects of the channel propertiesincluding voltage dependent gating, voltage dependent activation andinactivation, and most strikingly, increasing functional channel densityon the membrane. From molecular pharmacologic and electrophysiologicperspectives, there are more subtypes of voltage gated sodium channel inthe excitable cells than cloned α subunits. This may be partially due tothe existence of more α subunits in nature to be cloned andcharacterized. On the other hand, one might also expect that there mightbe more than one type of β1 and β2 subunits. In other words, the varietyof voltage gated sodium channel may result from the different types of αsubunit associating with one type of β1 and β2 subunits, and vice versa.In fact, biochemical study had revealed that there are more than onetype of sodium channel β1 subunit as determined by Western blot with β1specific antibody (Sutkowski, et al. 1990).

Recently β1A, a novel voltage-gated sodium channel subunit and splicevariant of β1, has been cloned and was reported to increase sodiumcurrent density at the plasma membrane and change voltage dependentkinetics when co-expressed with αIIA subunits in CHL fibroblasts(Kazan-Gillespie et al., 2000). β1A is developmentally regulated in thebrain. The subcellular distribution studies of rat VGSC β1A subunitdemonstrated that it was altered in addition to being up-regulated inthe DRG neurons as a consequence of peripheral nerve injury.Furthermore, rat VGSC β1A subunit may be important in the regulation ofthe aberrant array of sodium channels expressed subsequent to nerveinjury.

To study the human β1A subunit, we first tried to clone the subunitusing a homologous cloning strategy. Since β1A is an intron retainedsplicing variant at the carboxyl terminus of the β1 subunit, the forwardprimer was designed based on the human VGSC β1 subunit; while thereverse primer was designed based on rat VGSC β1A subunit. Unexpectedly,this pair of primers failed to amplify any DNA fragment from humanadrenal gland, fetal brain and adult brain Marathon™ ready cDNAlibraries. Three different reverse primers based on the sequenceencoding the carboxyl terminus of rat β1A subunit were then designed.However, none of these primers paired with forward primer could amplifyany DNA fragment from the above cDNA libraries under several PCRconditions, suggesting that human VGSC β1A subunit was significantlydifferent from its counterpart in rat.

In order to clone the splicing variant of human VGSC β1A subunit, aRapid Amplification of cDNA End (RACE-PCR) technique was used. Unlikeregular RT-PCR that requires two gene specific primers, RACE-PCRrequires only one specific primer pairing with a universal primer (AP1or AP2) for RT-PCR amplification. This requires adding an adaptorrecognized by the universal primer to the end of each cDNA when thelibrary was made. Currently, this type of cDNA library is commerciallyavailable (Marathon™ ready cDNA library, Clontech). Therefore, by thistechnique, human VGSC β1A subunit could be amplified with a human β1specific primer (SB1-10, see example 1) without knowing the sequence ofhuman β1A carboxyl terminus. The technical difficulty of thisapplication is to effectively distinguish novel β1A from the β1 subunitbecause of using β1 subunit specific primer for RACE-PCR. To solve thisproblem, the transformants were pre-screened by PCR with a pair ofprimers recognizing only the human VGSC β1 subunit and the negativeclones (which could not be amplified by such PCR) would be subjected forfurther characterization and sequencing. With this strategy, a novelhuman VGSC β1A subunit was cloned from human adrenal gland Marathon™ready cDNA library, and subsequently amplified from human fetal brainMarathon™ ready cDNA library with human β1A specific primers.

Analysis of the primary sequence revealed that the human VGSC β1Asubunit is also a splicing variant of the β1 subunit with a retainedintron and in frame stop codon. This novel VGSC β subunit also containsthe basic structure of VGSC β subunit: an amino-terminal extracellularimmunoglobulin-like motif and a carboxyl-terminal transmembrane domain.However, the human VGSC β1A subunit is significantly different from itsrat counterpart. They share only about 35% identity at their carboxylterminal coding region.

The present invention relates to DNA encoding human VGSC β1A subunitthat was isolated from human VGSC β1A subunit producing cells. The term“Human VGSC β1A subunit”, as used herein, refers to protein which canspecifically function as a channel subunit. That is, it can combine withthe other protein subunits to form a functioning calcium channel.

The recombinant protein is useful to identify modulators of thefunctional human VGSC β1A subunit. Northern blot analysis demonstratedthat the VGSC β1A subunit was widely distributed in a variety tissuesincluding, but not limited, in brain, heart, skeletal muscle, liver,lung, placenta, kidney and pancreas. In brain, the VGSC β1A subunitexpresses most highly in the cerebellum region. Immunohistochemicalstudy also demonstrates that the VGSC β1A was not only expressed indorsal root ganglia (DRG), but is also up-regulated after nerve injury,suggesting it plays a role in neuropathic pain. Alteration in sodiumchannel expression and/or function can have a profound influence on thefiring properties of peripheral and central neurons, and many othertissues. Modulators of VGSC β1A can be identified in the assays of thisinvention and tested for their use as therapeutic agents for neuropathicpain, chronic pain, febrile seizures and general epilepsy, localanesthetics, antiarrhythmics and anticonvulsants as well as many otherhuman diseases related to sodium channels (Wallace, et al. 1998,Porreca, et al, 1999, Balser 1999). Human VGSC β1A may also be useful inhuman diseases where other β1 sodium channel alterations are linked toaberrant sodium channel activity, such as generalized epilepsy withfebrile seizures plus (GEFS+) and congenital long-QT syndrome (LQT) (acardiac arrhythmia characterized in part by prolonged ventricularrepolarization).

Moreover, as provided in Example 12 below, the expression of the β1Asubunit and β1 was investigated in DRG neurons from a nerve ligationmodel of neuropathic pain in rats using immunohistochemistry and imageanalysis. The levels of β1A subunit and β1 expression were increasedmost notably in the nociceptive DRG neurons, although there wereincreases in the sensory DRG neurons as well. However, the subcellularlabeling of these two polypeptides differed dramatically in the DRGneurons subsequent to peripheral nerve injury. These studiesdemonstrated that peripheral nerve injury is associated withupregulation of β1A subunit and β1 sodium channel subunits and alteredcellular distribution patterns of β1A in DRG. This is evidence that thesodium channel β1A subunit is important in the regulation of theaberrant sodium channel expression in DRG neurons subsequent to nerveinjury.

Thus, this invention also contemplates the use of screening assaysemploying the human β1A subunit to identify modulators capable ofbinding to the β1A subunit. The modulators that are capable of bindingto the human β1A subunit can then be used as a therapeutic, such as in apharmaceutical composition, for the treatment of or as a method fordecreasing neuropathic pain in a human. The pharmaceutical compositionscomprising the modulator of the human β1A subunit are delivered to cellsbinding to a sodium channel β1A subunit-expressing cell in the human andfunction to decrease neuropathic pain.

A variety of cells and cell lines may be used to isolate Human β1Asodium channel subunit cDNA using primers selected based on thenucleotide sequence encoding the human β1A sodium channel subunit.

Any of a variety of procedures known in the art may be used tomolecularly clone Human β1A sodium channel subunit DNA to obtain relatedsequences or allelic variants. These methods include, but are notlimited to, direct functional expression of the Human β1A sodium channelsubunit genes following the construction of a Human β1A sodium channelsubunit-containing cDNA library in an appropriate expression vectorsystem. Another method is to screen Human β1A sodium channelsubunit-containing cDNA library constructed in a bacteriophage orplasmid shuttle vector with a labeled oligonucleotide probe designedfrom the amino acid sequence of the Human β1A sodium channel subunitsubunits. An additional method consists of screening a Human β1A sodiumchannel subunit-containing cDNA library constructed in a bacteriophageor plasmid shuttle vector with a partial cDNA encoding the Human β1Asodium channel subunit protein. This partial cDNA is obtained by thespecific PCR amplification of Human β1A sodium channel subunit DNAfragments through the design of degenerate oligonucleotide primers fromthe amino acid sequence of the purified Human β1A sodium channel subunitprotein.

Another method is to isolate RNA from human VGSC β1A subunit-producingcells and translate the RNA into protein via an in vitro or an in vivotranslation system. The translation of the RNA into a peptide a proteinwill result in the production of at least a portion of the Human β1Asodium channel subunit protein which can be identified by, for example,immunological reactivity with an anti-human β1A sodium channel subunitantibody or by biological activity of Human β1A sodium channel subunitprotein. In this method, pools of RNA isolated from Human β1A sodiumchannel subunit-producing cells can be analyzed for the presence of anRNA that encodes at least a portion of the Human β1A sodium channelsubunit protein. Further fractionation of the RNA pool results inpurification of the Human β1A sodium channel subunit RNA from non-Humanβ1A sodium channel subunit RNA. The peptide or protein produced by thismethod may be analyzed to provide amino acid sequences which in turn areused to provide primers for production of Human β1A sodium channelsubunit cDNA, or the RNA used for translation can be analyzed to providenucleotide sequences encoding Human β1A sodium channel subunit andproduce probes for this production of Human β1A sodium channel subunitcDNA. This method is known in the art and can be found in, for example,Maniatis, T., Fritsch, E. F., Sambrook, J. in Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. 1989.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have Human β1Asodium channel subunit activity. The selection of cells or cell linesfor use in preparing a cDNA library to isolate Human β1A sodium channelsubunit cDNA may be done by first measuring cell associated Human β1Asodium channel subunit activity using the measurement of Human 01Asodium channel subunit-associated biological activity or a ligandbinding assay.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Maniatis, T., Fritsch, E. F., Sambrook, J.,Molecular Cloning: A Laboratory Manual, Second Edition (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989).

It is also readily apparent to those skilled in the art that DNAencoding Human β1A sodium channel subunit may also be isolated from asuitable genomic DNA library. Construction of genomic DNA libraries canbe performed by standard techniques well known in the art. Well knowngenomic DNA library construction techniques can be found in Maniatis,T., Fritsch, E. F., Sambrook, J. in Molecular Cloning: A LaboratoryManual, Second Edition (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

In order to clone the human sodium channel β1A subunit gene by the abovemethods, the amino acid sequence of Human sodium channel β1A subunit maybe necessary. To accomplish this, Human β1A sodium channel subunitprotein may be purified and partial amino acid sequence determined byautomated sequencers. It is not necessary to determine the entire aminoacid sequence, but the linear sequence of two regions of 6 to 8 aminoacids from the protein is determined for the production of primers forPCR amplification of a partial Human β1A sodium channel subunit DNAfragment.

Once suitable amino acid sequences have been identified, the DNAsequences capable of encoding them are synthesized. Because the geneticcode is degenerate, more than one codon may be used to encode aparticular amino acid, and therefore, the amino acid sequence can beencoded by any of a set of similar DNA oligonucleotides. Only one memberof the set will be identical to the Human β1A sodium channel subunitsequence but will be capable of hybridizing to Human β1A sodium channelsubunit DNA even in the presence of DNA oligonucleotides withmismatches. The mismatched DNA oligonucleotides may still sufficientlyhybridize to the Human sodium channel β1A subunit DNA to permitidentification and isolation of Human sodium channel β1A subunitencoding DNA. DNA isolated by these methods can be used to screen DNAlibraries from a variety of cell types, from invertebrate and vertebratesources, and to isolate homologous genes.

Purified biologically active Human sodium channel β1A subunit may haveseveral different physical forms. The Human sodium channel β1A subunitmay exist as a full-length nascent or unprocessed polypeptide, or aspartially processed polypeptides or combinations of processedpolypeptides. The full-length nascent Human β1A sodium channel subunitpolypeptide may be post-translationally modified by specific proteolyticcleavage events that results in the formation of fragments of the fulllength nascent polypeptide. A fragment or physical association offragments may have the full biological activity associated with Humanβ1A sodium channel subunit however, the degree of Human β1A sodiumchannel subunit activity may vary between individual Human β1A sodiumchannel subunit fragments and physically associated Human β1A sodiumchannel subunit polypeptide fragments.

Because the genetic code is degenerate, more than one codon may be usedto encode a particular amino acid, and therefore, the amino acidsequence can be encoded by any of a set of similar DNA oligonucleotides.Only one member of the set will be identical to the Human β1A sodiumchannel subunit sequence but will be capable of hybridizing to Human β1Asodium channel subunit DNA even in the presence of DNA oligonucleotideswith mismatches under appropriate conditions. Under alternateconditions, the mismatched DNA oligonucleotides may still hybridize tothe Human β1A sodium channel subunit DNA to permit identification andisolation of Human β1A sodium channel subunit encoding DNA.

DNA encoding Human β1A sodium channel subunit from a particular organismmay be used to isolate and purify homologues of Human β1A sodium channelsubunit from other organisms. To accomplish this, the first Human β1Asodium channel subunit DNA may be mixed with a sample containing DNAencoding homologues of Human β1A sodium channel subunit underappropriate hybridization conditions. The hybridized DNA complex may beisolated and the DNA encoding the homologous DNA may be purifiedtherefrom.

Functional Derivatives/Variants

It is known that there is a substantial amount of redundancy in thevarious codons that code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences that containalternative codons that code for the eventual translation of theidentical amino acid. For purposes of this specification, a sequencebearing one or more replaced codons will be defined as a degeneratevariation. Also included within the scope of this invention aremutations either in the DNA sequence or the translated protein, which donot substantially alter the ultimate physical properties of theexpressed protein. For example, substitution of aliphatic amino acidsAlanine, Valine, Leucine and Isoleucine; interchange of the hydroxylresidues Serine and Threonine, exchange of the acidic residues Asparticacid and Glutamic acid, substitution between the amide residuesAsparagine and Glutamine, exchange of the basic residues Lysine andArginine and human β1A sodium channel subunits among the aromaticresidues Phenylalanine, Tyrosine may not cause a change in functionalityof the polypeptide. Such substitutions are well known and are described,for instance in Molecular Biology of the Gene, 4^(th) Ed. BengaminCummings Pub. Co. by Watson et al.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude, but are not limited to site directed mutagenesis, chimericsubstitution, and gene fusions. Site-directed mutagenesis is used tochange one or more DNA residues that may result in a silent mutation, aconservative mutation, or a nonconservative mutation. Chimeric genes areprepared by swapping domains of similar or different genes to replacesimilar domains in the human β1A sodium channel subunit gene. Similarly,fusion genes may be prepared that add domains to the human β1A sodiumchannel subunit gene, such as an affinity tag to facilitateidentification and isolation of the gene. Fusion genes may be preparedto replace regions of the human β1A sodium channel subunit gene, forexample to create a soluble version of the protein by removing atransmembrane domain or adding a targeting sequence to redirect thenormal transport of the protein, or adding new post-translationalmodification sequences to the human β1A sodium channel subunit gene.Examples of altered properties include but are not limited to changes inthe affinity of an enzyme for a substrate or a receptor for a ligand.All such changes of the polynucleotide or polypeptide sequences areanticipated as useful variants of the present invention so long as theoriginal function of the polynucleotide or polypeptide sequence of thepresent invention is maintained as described herein.

IDENTITY or SIMILARITY, as known in the art, are relationships betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Both identityand similarity can be readily calculated (Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). While there exist a numberof methods to measure identity and similarity between two polynucleotideor two polypeptide sequences, both terms are well known to skilledartisans (Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H.,and Lipman, D., (1988) SIAM J. Applied Math., 48, 1073). Methodscommonly employed to determine identity or similarity between sequencesinclude, but are not limited to those disclosed in Carillo, H., andLipman, D., (1988) SIAM J. Applied Math., 48, 1073. Preferred methods todetermine identity are designed to give the largest match between thesequences tested. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux, J., et al., (1984)Nucleic Acids Research 12(1), 387), BLASTP, BLASTN, and FASTA (Atschul,S. F. et al., (1990) J. Molec. Biol. 215, 403).

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single- and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions or single-, double- andtriple-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded, or a mixture of single- anddouble-stranded regions. In addition, polynucleotide as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term polynucleotide includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotides” as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The termpolynucleotide as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including simple and complex cells, inter alia. Polynucleotidesembraces short polynucleotides often referred to as oligonucleotide(s).

The term polypeptides, as used herein, refers to the basic chemicalstructure of polypeptides that is well known and has been described ininnumerable textbooks and other publications in the art. In thiscontext, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types. It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, may be modified in a given polypeptide, eitherby natural processes, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art.

Among the known modifications which may be present in polypeptides ofthe present are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known to those of skill and have been described in great detailin the scientific literature. Several particularly common modifications,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation, forinstance, are described in most basic texts, such as, for instancePROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W.H. Freeman and Company, New York (1993). Many detailed reviews areavailable on this subject, such as, for example, those provided by Wold,F., Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., (1990)Meth. Enzymol. 182, 626-646 and Rattan et al., “Protein Synthesis:Posttranslational Modifications and Aging”, (1992) Ann. N.Y. Acad. Sci.663, 48-62.

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be generally as a result of posttranslational events, includingnatural processing event and events brought about by human manipulationwhich do not occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well. Modifications can occur anywherein a polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. In fact, blockage of theamino or carboxyl group in a polypeptide, or both, by a covalentmodification, is common in naturally occurring and syntheticpolypeptides and such modifications may be present in polypeptides ofthe present invention, as well. For instance, the amino terminal residueof polypeptides made in E. coli or other cells, prior to proteolyticprocessing, almost invariably will be N-formylmethionine. Duringpost-translational modification of the peptide, a methionine residue atthe NH.sub.2-terminus may be deleted. Accordingly, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of the protein of the invention.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as, forexample, E. coli. Accordingly, when glycosylation is desired, apolypeptide should be expressed in a glycosylating host, generally aeukaryotic cell. Insect cells often carry out the same posttranslationalglycosylations as mammalian cells and, for this reason, insect cellexpression systems have been developed to efficiently express mammalianproteins having native patterns of glycosylation, inter alia. Similarconsiderations apply to other modifications. It will be appreciated thatthe same type of modification may be present in the same or varyingdegree at several sites in a given polypeptide. Also, a givenpolypeptide may contain many types of modifications. In general, as usedherein, the term polypeptide encompasses all such modifications,particularly those that are present in polypeptides synthesizedrecombinantly by expressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. A variant of thepolynucleotide may be a naturally occurring variant such as a naturallyoccurring allelic variant, or it may be a variant that is not known tooccur naturally. A polynucleotide variant is a polynucleotide thatdiffers in nucleotide sequence from another, reference polynucleotide.Generally, differences are limited so that the nucleotide sequences ofthe reference and the variant are closely similar overall and, in manyregions, identical. As noted below, changes in the nucleotide sequenceof the variant may be silent. That is, they may not alter the aminoacids encoded by the polynucleotide. Where alterations are limited tosilent changes of this type a variant will encode a polypeptide with thesame amino acid sequence as the reference. Also as noted below, changesin the nucleotide sequence of the variant may alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide. Suchnucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed above.

A polypeptide variant is a polypeptide that differs in amino acidsequence from another, reference polypeptide. Generally, differences arelimited so that the sequences of the reference and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions, fusions and truncations, which maybe present in any combination. As used herein, a “functional derivative”of Human β1A sodium channel subunit is a compound that possesses abiological activity (either functional or structural) that issubstantially similar to the biological activity of Human β1A sodiumchannel subunit. The term “functional derivatives” is intended toinclude the “fragments,” “variants,” “degenerate variants,” “analogs”and “homologues” or to “chemical derivatives” of Human β1A sodiumchannel subunit. Useful chemical derivatives of polypeptide are wellknown in the art and include, for example, covalent modification of oneor more reactive organic sites contained within the polypeptide with asecondary chemical moiety. Well known cross-linking reagents are usefulto react to amino, carboxyl, or aldehyde residues to introduce, forexample, an affinity tag such as biotin, a fluorescent dye, or toconjugate the polypeptide to a solid phase surface (for example tocreate an affinity resin). The term “fragment” is meant to refer to anypolypeptide subset of the Human β1A sodium channel subunit.

A molecule is “substantially similar” to a Human β1A sodium channelsubunit if both molecules have substantially similar structures or ifboth molecules possess similar biological activity. Therefore, if thetwo molecules possess substantially similar activity, they areconsidered to be variants even if the structure of one of the moleculesis not found in the other or even if the two amino acid sequences arenot identical. The term “analog” refers to a molecule substantiallysimilar in function to either the entire Human β1A sodium channelsubunit molecule or to a fragment thereof. Further particularlypreferred in this regard are polynucleotides encoding variants, analogs,derivatives and fragments of SEQ.ID.NO.:13, and variants, analogs andderivatives of the fragments, which have the amino acid sequence of thepolypeptide of SEQ.ID.NO.:14 in which several, a few, 5 to 10, 1 to 5, 1to 3, 2, 1 or no amino acid residues are substituted, deleted or added,in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the gene of SEQ.ID.NO.:13. Also especiallypreferred in this regard are conservative substitutions. Most highlypreferred are polynucleotides encoding polypeptides having the aminoacid sequence of SEQ.ID.NO.:14, without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 75% identical over their entire length to a polynucleotideencoding the polypeptide having the amino acid sequence set out inSEQ.ID.NO.:14, and polynucleotides which are complementary to suchpolynucleotides. Yet other preferred embodiments of the invention arepolynucleotides that are at least 75% identical over a consecutiveportion of their length to a polynucleotide encoding the polypeptidehaving the amino acid sequence 150 to 268 set out in SEQ.ID.NO.:14, andpolynucleotides which are complementary to such polynucleotides.Alternatively, highly preferred are polynucleotides that comprise aregion that is at least 80% identical, more highly preferred arepolynucleotides at comprise a region that is at least 90% identical, andamong these preferred polynucleotides, those with at least 95% areespecially preferred. Furthermore, those with at least 97% identity arehighly preferred among those with at least 95%, and among these thosewith at least 98% and at least 99% are particularly highly preferred,with at least 99% being the most preferred. The polynucleotides, whichhybridize to the polynucleotides described herein, in a preferredembodiment, encode polypeptides, which retain substantially the samebiological function or activity as the polypeptide characterized by thededuced amino acid sequence of SEQ.ID.NO.:14. Preferred embodiments inthis respect, moreover, are polynucleotides that encode polypeptidesthat retain substantially the same biological function or activity asthe mature polypeptide encoded by the DNA of SEQ.ID.NO.:13. The presentinvention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the present inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the term “stringent conditions” means hybridization will occuronly if there is at least 95% and preferably at least 97% identitybetween the sequences.

There are a large numbers of polynucleotide hybridization techniquesknown in the art including hybridizations coupling DNA to DNA, RNA toRNA and RNA to DNA. All of these methods can incorporate stringenthybridization conditions to facilitate the accurate identification ofnucleic acid targeting to a hybridizable probe. As is known in the art,methods vary depending on the substrate used for hybridization andManiatis et al. supra, as well as a variety of references in the artdetail a number of stringent hybridization techniques. In one example,DNA or RNA samples to be probed are immobilized on a suitable substratesuch as nitrocellulose, nylon, polyvinylidene difluoride, or the like. Apurified probe, preferably with sufficient specific activity (generallygreater than about 10⁸ cpm/μg probe), substantially free ofcontaminating DNA, protein or unincorporated nucleotides is used. Wherenitrocellulose is used, and the immobilized nucleic acid is DNAimmobilized on nitrocellulose, the nitrocellulose with DNA is incubatedwith a hybridization solution comprising 50% formamide-deionized, 6×SSC,1% SDS, 0.1% Tween 20 and 100 μg/ml t RNA at 42° C. for 15 minutes.Probe is added and the nitrocellulose is further immobilized at 42° C.for about 12-19 hours. The nitrocellulose is then washed in at least twosuccessive washes at 22° C. followed by stringent washes at 65° C. in abuffer of 0.04M sodium phosphate, pH 7.2, 1% SDS and 1 mM EDTA.Conditions for increasing the stringency of a variety of nucleotidehybridizations are well known in the art.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention may be used asa hybridization probe for RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding the sequences ofSEQ.ID.NO.:13 and to isolate cDNA and genomic clones of other genes thathave a high sequence similarity to SEQ.ID.NO.:13. Such probes generallywill comprise at least 15 bases. Preferably, such probes will have atleast 30 bases and may have at least 50 bases. Particularly preferredprobes will have at least 30 bases and will have 50 bases or less. Forexample, the coding region of the gene of the invention may be isolatedby screening using the known DNA sequence to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the present invention is then used toscreen a library of cDNA, genomic DNA or mRNA to determine to whichmembers of the library the probe hybridizes.

The polypeptides of the present invention include the polypeptide ofSEQ.ID.NO.:14 (in particular the mature polypeptide) as well aspolypeptides which have at least 75% identity to the polypeptide ofSEQ.ID.NO.:14, preferably at least 80% identity to the polypeptide ofSEQ.ID.NO.:14, and more preferably at least 90% similarity (morepreferably at least 90% identity) to the polypeptide of SEQ.ID.NO.:14and still more preferably at least 95% similarity (still more preferablyat least 95% identity) to the polypeptide of SEQ.ID.NO.:14 and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids. Representative examples ofpolypeptide fragments of the invention, include, for example, truncationpolypeptides of SEQ.ID.NO.:14.

Truncation polypeptides include polypeptides having the amino acidsequence of SEQ.ID.NO.:14, or of variants or derivatives thereof, exceptfor deletion of a continuous series of residues (that is, a continuousregion, part or portion) that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus or, asin double truncation mutants, deletion of two continuous series ofresidues, one including the amino terminus and one including thecarboxyl terminus. Also preferred in this aspect of the invention arefragments characterized by structural or functional attributes of thepolypeptide characterized by the sequences of SEQ.ID.NO.:14. Preferredembodiments of the invention in this regard include fragments thatcomprise α-helix and α-helix forming regions, β-sheet andβ-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, αamphipathic regions, β amphipathic regions, flexible regions,surface-forming regions, substrate binding region, high antigenic indexregions of the polypeptide of the invention, and combinations of suchfragments. Preferred regions are those that mediate activities of thepolypeptides of the invention. Most highly preferred in this regard arefragments that have a chemical, biological or other activity of theresponse regulator polypeptide of the invention, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity.

Recombinant Expression of Human β1A Sodium Channel Subunit

The cloned Human β1A sodium channel subunit DNA obtained through themethods described herein may be recombinantly expressed by molecularcloning into an expression vector containing a suitable promoter andother appropriate transcription regulatory elements, and transferredinto prokaryotic or eukaryotic host cells to produce recombinant Humanβ1A sodium channel subunit protein. Techniques for such manipulationsare fully described in Maniatis, T. et al., supra, and are well known inthe art.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned copies of genes and the translation oftheir mRNAs in an appropriate host. Such vectors can be used to expresseukaryotic genes in a variety of hosts such as bacteria including E.coli, bluegreen algae, plant cells, insect cells, fungal cells includingyeast cells, and animal cells.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells or bacteria-fungal cellsor bacteria-invertebrate cells. An appropriately constructed expressionvector should contain: an origin of replication for autonomousreplication in host cells, selectable markers, a limited number ofuseful restriction enzyme sites, a potential for high copy number, andactive promoters. A promoter is defined as a DNA sequence that directsRNA polymerase to bind to DNA and initiate RNA synthesis. A strongpromoter is one that causes mRNAs to be initiated at high frequency.Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.

A variety of mammalian expression vectors may be used to expressrecombinant Human β1A sodium channel subunit in mammalian cells.Commercially available mammalian expression vectors which may besuitable for recombinant Human β1A sodium channel subunit expression,include but are not limited to, pMAMneo (Clontech), pIRES vectors(Clontech), pTET-On and pTET-Off (Clontech), pcDNA3 (Invitrogen),pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo(ATCC 37593, ATCC, Manassas, Va.) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198),pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC 37565).

A variety of bacterial expression vectors may be used to expressrecombinant Human β1A sodium channel subunit in bacterial cells.Commercially available bacterial expression vectors which may besuitable for recombinant Human β1A sodium channel subunit expressioninclude, but are not limited to, pET vectors (Novagen), pGEX vectors(Pharmacia) and pQE vectors (Qiagen).

A variety of fungal cell expression vectors may be used to expressrecombinant Human β1A sodium channel subunit in fungal cells such asyeast. Commercially available fungal cell expression vectors which maybe suitable for recombinant Human β1A sodium channel subunit expressioninclude but are not limited to pYES2 (Invitrogen) and Pichia expressionvector (Invitrogen).

A variety of insect cell expression vectors may be used to expressrecombinant Human β1A sodium channel subunit in insect cells.Commercially available insect cell expression vectors which may besuitable for recombinant expression of Human β1A sodium channel subunitinclude, but are not limited to, pBlueBacHII (Invitrogen).

DNA encoding Human β1A sodium channel subunit may be cloned into anexpression vector for expression in a recombinant host cell. Recombinanthost cells may be prokaryotic or eukaryotic, including, but not limitedto, bacteria such as E. coli, fungal cells such as yeast, mammaliancells including, but not limited to, cell lines of human, bovine,porcine, monkey and rodent origin, and insect cells including, but notlimited to, drosophila and silkworm derived cell lines. Cell linesderived from mammalian species which may be suitable and which arecommercially available, include, but are not limited to, CV-1 (ATCC CCL70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61),3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I(ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), L-cells,HEK-293 (ATCC CRL1573), PC12 (ATCC CRL-1721).

The expression vector may be introduced into host cells via any one of anumber of techniques including, but not limited to, transformation,transfection, protoplast fusion, lipofection, and electroporation. Theexpression vector-containing cells are clonally propagated andindividually analyzed to determine whether they produce Human sodiumchannel β1A subunit protein. Identification of Human β1A sodium channelsubunit expressing host cell clones may be done by several means,including but not limited to immunological reactivity with anti-humanβ1A sodium channel subunit antibodies, and the presence of hostcell-associated Human β1A sodium channel subunit activity.

Expression of Human sodium channel β1A subunit DNA may also be performedusing in vitro produced synthetic mRNA. Synthetic mRNA or mRNA isolatedfrom Human β1A sodium channel subunit producing cells can be efficientlytranslated in various cell-free systems including, but not limited to,wheat germ extracts and reticulocyte extracts, as well as efficientlytranslated in cell based systems including, but not limited to,microinjection into frog oocytes, with microinjection into frog oocytesbeing generally preferred.

To determine the Human β1A sodium channel subunit DNA sequence(s) thatyields optimal levels of Human β1A sodium channel subunit activityand/or Human β1A sodium channel subunit protein, Human β1A sodiumchannel subunit DNA molecules including, but not limited to, thefollowing can be constructed: the full-length open reading frame of theHuman β1A sodium channel subunit cDNA encoding the 32 kDa protein fromapproximately base 4 to approximately base 808 (these numbers correspondto first nucleotide of first methionine and last nucleotide before thefirst stop codon) and several constructs containing portions of the cDNAencoding Human β1A sodium channel subunit protein. All constructs can bedesigned to contain none, all or portions of the 5′ or the 3′untranslated region of Human β1A sodium channel subunit cDNA. Human β1Asodium channel subunit activity and levels of protein expression can bedetermined following the introduction, both singly and in combination,of these constructs into appropriate host cells. Following determinationof the Human β1A sodium channel subunit DNA cassette yielding optimalexpression in transient assays, this Human β1A sodium channel subunitDNA construct is transferred to a variety of expression vectors, forexpression in host cells including, but not limited to, mammalian cells,baculovirus-infected insect cells, E. coli, and the yeast S. cerevisiae.

Assay Methods for Human β1A Sodium Channel Subunit

Host cell transfectants and microinjected oocytes may be used to assayboth the levels of functional Human β1A sodium channel subunit activityand levels of total Human β1A sodium channel subunit protein by thefollowing methods. In the case of recombinant host cells, this involvesthe co-transfection of one or possibly two or more plasmids, containingthe Human sodium channel β1A subunit DNA encoding one or more fragmentsor subunits. In the case of oocytes, this involves the co-injection ofsynthetic RNAs for Human sodium channel β1A subunit protein. Followingan appropriate period of time to allow for expression, cellular proteinis metabolically labeled with, for example ³⁵S-methionine for 24 hours,after which cell lysates and cell culture supernatants are harvested andsubjected to immunoprecipitation with polyclonal antibodies directedagainst the Human β1A sodium channel subunit protein.

Levels of Human β1A sodium channel subunit protein in host cells arequantitated by immunoaffinity and/or ligand affinity techniques. Cellsexpressing Human β1A sodium channel subunit can be assayed for thenumber of Human β1A sodium channel subunit molecules expressed bymeasuring the amount of radioactive [ligand] binding to cell membranes.Human β1A sodium channel subunit-specific affinity beads or Human β1Asodium channel subunit-specific antibodies are used to isolate forexample ³⁵S-methionine labeled or unlabelled Human β1A sodium channelsubunit protein. Labeled Human β1A sodium channel subunit protein isanalyzed by SDS-PAGE. Unlabelled Human β1A sodium channel subunitprotein is detected by Western blotting, ELISA or RIA assays employingHuman β1A sodium channel subunit specific antibodies.

Other methods for detecting Human β1A sodium channel subunit activityinvolve the direct measurement of Human β1A sodium channel subunitactivity in whole cells transfected with Human β1A sodium channelsubunit cDNA or oocytes injected with Human β1A sodium channel subunitmRNA and optionally a sodium channel subunit mRNA. Human β1A sodiumchannel subunit activity is measured by biological characteristics ofthe host cells expressing Human β1A sodium channel subunit DNA. In thecase of recombinant host cells expressing Human β1A sodium channelsubunit patch voltage clamp techniques can be used to measure channelactivity and quantify modification of a sodium channel subunit ion fluxas a function of Human β1A sodium channel subunit protein. In the caseof oocytes patch clamp as well as two-electrode voltage clamp techniquescan be used to measure sodium channel activity and quantify Human β1Asodium channel subunit protein.

Cell Based Assays

The present invention provides a whole cell or isolated cell membranemethod to detect compound modulation of human β1A sodium channelsubunit. The method comprises the steps;

1) contacting a compound, and a cell or isolated cell membrane thatcontains functional human β1A sodium channel subunit, and

2) measuring a change in the cell or isolated cell membrane in responseto modified human β1A sodium channel subunit function by the compound.

The amount of time necessary for cell or cell membrane contact with thecompound is empirically determined, for example, by running a timecourse with a known human β1A sodium channel subunit modulator andmeasuring cellular changes as a function of time.

The measurement means of the method of the present invention can befurther defined by comparing a cell or cell membrane that has beenexposed to a compound to an identical cell or cell membrane preparationthat has not been similarly expose to the compound. Alternatively twocells, one containing functional human β1A sodium channel subunit and asecond cell identical to the first, but lacking functional human β1Asodium channel subunit could be both used. Both cells or cell membranesare contacted with the same compound and compared for differencesbetween the two cells. This technique is also useful in establishing thebackground noise of these assays. One of average skill in the art willappreciate that these control mechanisms also allow easy selection ofcellular changes that are responsive to modulation of functional humanβ1A sodium channel subunit.

Particularly preferred cell based assays (or cell membrane assays, ifsuitable) are those where the cell expresses an endogenous orrecombinant sodium α channel subunit simultaneously with recombinanthuman β1A. In these assays, a putative modulating compound can beanalyzed for its effect on electrophysiological changes to the sodiumflux upon the cell for altered expression of β1A expression, or alteredexpression of the α/β1A complex. Cells expressing recombinant human β1Aare subjected to electrophysiological analysis to measure the totalinflux of sodium ions (Na⁺) across the cell membrane by way of voltagedifferential using techniques well known by artisans in the field anddescribed herein, including patch clamp voltage techniques as well asmembrane proximal voltage sensitive dyes. Compounds that affect theproper function of human β1 may increase or decrease the capacity toopen the Na channel, may increase or decrease the rate of Na influx(thus affect the change of membrane potential), may increase or decreasethe rate of desensitization or re-sensitization of the channel. The term“test compound” or “modulating compound” as used herein in connectionwith a suspected modulator of human β1A refers to an organic moleculethat has the potential to disrupt specific ion channel activity or cellsurface expression of human β1A. For example, but not to limit the scopeof the current invention, compounds may include small organic molecules,synthetic or natural amino acid peptides, proteins, or synthetic ornatural nucleic acid sequences, or any chemical derivatives of theaforementioned.

The term “cell” refers to at least one cell, but includes a plurality ofcells appropriate for the sensitivity of the detection method. Cellssuitable for the present invention may be bacterial, yeast, oreukaryotic. For assays to which electrophysiological analysis isconducted, the cells must be eukaryotic, preferably selected from agroup consisting of Xenopus oocytes, or PC12, COS-7, CHO, HEK293,SK-N-SH cells.

The assay methods to determine compound modulation of functional humansodium channel β1A subunit can be in conventional laboratory format oradapted for high throughput. The term “high throughput” refers to anassay design that allows easy analysis of multiple samplessimultaneously, and capacity for robotic manipulation. Another desiredfeature of high throughput assays is an assay design that is optimizedto reduce reagent usage, or minimize the number of manipulations inorder to achieve the analysis desired. Examples of assay formats include96-well or 384-well plates, levitating droplets, and “lab on a chip”microchannel chips used for liquid handling experiments. It is wellknown by those in the art that as miniaturization of plastic molds andliquid handling devices are advanced, or as improved assay devices aredesigned, that greater numbers of samples may be performed using thedesign of the present invention.

The cellular changes suitable for the method of the present inventioncomprise directly measuring changes in the function or quantity of humanβ1A sodium channel subunit, or by measuring downstream effects of humanβ1A sodium channel subunit function, for example by measuring secondarymessenger concentrations or changes in transcription or by changes inprotein levels of genes that are transcriptionally influenced by humanβ1A sodium channel subunit, or by measuring phenotypic changes in thecell. Preferred measurement means include changes in the quantity ofhuman β1A sodium channel subunit protein, changes in the functionalactivity of human β1A sodium channel subunit, changes in the quantity ofmRNA, changes in intracellular protein, changes in cell surface protein,or secreted protein, or changes in Ca+2, cAMP or GTP concentration.Changes in the quantity or functional activity of human β1A sodiumchannel subunit are described herein. Changes in the levels of mRNA aredetected by reverse transcription polymerase chain reaction (RT-PCR) orby differential gene expression. Immunoaffinity, ligand affinity, orenzymatic measurement quantitated changes in levels of protein in hostcells. Protein-specific affinity beads or specific antibodies are usedto isolate for example ³⁵S-methionine labeled or unlabelled protein.Labeled protein is analyzed by SDS-PAGE. Unlabelled protein is detectedby Western blotting, cell surface detection by fluorescent cell sorting,cell image analysis, ELISA or RIA employing specific antibodies. Wherethe protein is an enzyme, the induction of protein is monitored bycleavage of a fluorogenic or colorimetric substrate.

Preferred detection means for cell surface protein include flowcytometry or statistical cell imaging. In both techniques the protein ofinterest is localized at the cell surface, labeled with a specificfluorescent probe, and detected via the degree of cellular fluorescence.In flow cytometry, the cells are analyzed in a solution, whereas incellular imaging techniques, a field of cells is compared for relativefluorescence.

A preferred detection means for secreted proteins that are enzymes suchas alkaline phosphatase or proteases, would be fluorescent orcolorimetric enzymatic assays. Fluorescent/luminescent/color substratesfor alkaline phosphatase are commercially available and such assays areeasily adaptable to high throughput multiwell plate screen format.Fluorescent energy transfer based assays are used for protease assays.Fluorophore and quencher molecules are incorporated into the two ends ofthe peptide substrate of the protease. Upon cleavage of the specificsubstrate, separation of the fluorophore and quencher allows thefluorescence to be detectable. When the secreted protein could bemeasured by radioactive methods, scintillation proximity technologycould be used. The substrate of the protein of interest is immobilizedeither by coating or incorporation on a solid support that contains afluorescent material. A radioactive molecule, brought in close proximityto the solid phase by enzyme reaction, causes the fluorescent materialto become excited and emit visible light. Emission of visible lightforms the basis of detection of successful ligand/target interaction,and is measured by an appropriate monitoring device. An example of ascintillation proximity assay is disclosed in U.S. Pat. No. 4,568,649,issued Feb. 4, 1986. Materials for these types of assays arecommercially available from Dupont NEN® (Boston, Mass.) under the tradename FlashPlate™.

A preferred detection means where the endogenous gene results inphenotypic cellular structural changes is statistical image analysis thecellular morphology or intracellular phenotypic changes. For example,but not by way of limitation, a cell may change morphology such arounding versus remaining flat against a surface, or may becomegrowth-surface independent and thus resemble transformed cell phenotypewell known in the art of tumor cell biology, or a cell may produce newoutgrowths. Phenotypic changes that may occur intracellularly includecytoskeletal changes, alteration in the endoplasmic reticulum/Golgicomplex in response to new gene transcription, or production of newvesicles.

Where the endogenous gene encodes a soluble intracellular protein,changes in the endogenous gene may be measured by changes of thespecific protein contained within the cell lysate. The soluble proteinmay be measured by the methods described herein.

The present invention is also directed to methods for screening forcompounds that modulate the expression of DNA or RNA encoding Human β1Asodium channel subunit as well as the function of Human β1A sodiumchannel subunit protein in vivo. Compounds may modulate by increasing orattenuating the expression of DNA or RNA encoding a Human β1A sodiumchannel subunit, or the function of a Human β1A sodium channel subunitprotein. Compounds that modulate the expression of DNA or RNA encoding aHuman β1A sodium channel subunit or the function of a Human β1A sodiumchannel subunit protein may be detected by a variety of assays. Theassay may be a simple “yes/no” assay to determine whether there is achange in expression or function. The assay may be made quantitative bycomparing the expression or function of a test sample with the levels ofexpression or function in a standard sample. Modulators identified inthis process are useful as candidate therapeutic agents.

Purification of Human β1A Sodium Channel Subunit Protein

Following expression of the Human β1A sodium channel subunit in arecombinant host cell, the Human β1A sodium channel subunit protein maybe recovered to provide the purified Human β1A sodium channel subunit inactive form. Several Human β1A sodium channel subunit purificationprocedures are available and suitable for use. As described above forpurification of Human β1A sodium channel subunit from natural sources arecombinant Human β1A sodium channel subunit may be purified from celllysates and extracts, or from conditioned culture medium, by variouscombinations of, or individual application of salt fractionation, ionexchange chromatography, size exclusion chromatography, hydroxylapatiteadsorption chromatography and hydrophobic interaction chromatography,lectin chromatography, and antibody/ligand affinity chromatography.

Recombinant Human sodium channel β1A subunits can be separated fromother cellular proteins through the use of an immunoaffinity column madewith monoclonal or polyclonal antibodies specific for full lengthnascent Human β1A sodium channel subunit, polypeptide fragments of Humanβ1A sodium channel subunit or Human β1A sodium channel subunit subunits.The affinity resin is then equilibrated in a suitable buffer, forexample phosphate buffered saline (pH 7.3), and the cell culturesupernatants or cell extracts containing a Human β1A sodium channelsubunit or Human β1A sodium channel subunit subunits are slowly passedthrough the column. The column is then washed with the buffer until theoptical density (A₂₈₀) falls to background, then the protein is elutedby changing the buffer condition, such as by lowering the pH using abuffer such as 0.23 M glycine-HCl (pH 2.6). The purified Human sodiumchannel β1A subunit protein is then dialyzed against a suitable buffersuch as phosphate buffered saline.

Protein Based Assay

The present invention provides an in vitro protein assay method todetect compound modulation of human sodium channel β1A subunit proteinactivity. The method comprises the steps;

1) contacting a compound and a human β1A sodium channel subunit protein,and

2) measuring a change to human β1A sodium channel subunit function bythe compound.

The amount of time necessary for cellular contact with the compound isempirically determined, for example, by running a time course with aknown human sodium channel β1A subunit modulator and measuring changesas a function of time.

Production and Use of Antibodies that Bind to Human β1A Sodium ChannelSubunit

Monospecific antibodies to Human β1A sodium channel subunit are purifiedfrom mammalian antisera containing antibodies reactive against Human β1Asodium channel subunit or are prepared as monoclonal antibodies reactivewith Human β1A sodium channel subunit using the technique originallydescribed by Kohler and Milstein, Nature 256: 495-497 (1975).Immunological techniques are well known in the art and described in, forexample, Antibodies: A laboratory manual published by Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., ISBN 0879693142.Monospecific antibody as used herein is defined as a single antibodyspecies or multiple antibody species with homogenous bindingcharacteristics for the human sodium channel β1A subunit. “Homogenousbinding” as used herein refers to the ability of the antibody species tobind to a specific antigen or epitope, such as those associated with theHuman β1A sodium channel subunit, as described above. Human β1A sodiumchannel subunit specific antibodies are raised by immunizing animalssuch as mice, rats, guinea pigs, rabbits, goats, horses and the like,with rabbits being preferred, with an appropriate concentration of Humanβ1A sodium channel subunit either with or without an immune adjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.001 mg and about 1000 mg of human sodiumchannel β1A subunit associated with an acceptable immune adjuvant. Suchacceptable adjuvants include, but are not limited to, Freund's complete,Freund's incomplete; alum-precipitate, water in oil emulsion containingCorynebacterium parvum and tRNA. The initial immunization consists ofhuman sodium channel β1A subunit in, preferably, Freund's completeadjuvant at multiple sites either subcutaneously (SC), intraperitoneally(IP) or both. Each animal is bled at regular intervals, preferablyweekly, to determine antibody titer. The animals may or may not receivebooster injections following the initial immunization. Those animalsreceiving booster injections are generally given an equal amount of theantigen in Freund's incomplete adjuvant by the same route. Boosterinjections are given at about three-week intervals until maximal titersare obtained. At about 7 days after each booster immunization or aboutweekly after a single immunization, the animals are bled, the serumcollected, and aliquots are stored at about −20° C.

Monoclonal antibodies (mAb) reactive with Human β1A sodium channelsubunit are prepared by immunizing inbred mice, preferably Balb/c, withHuman β1A sodium channel subunit. The mice are immunized by the IP or SCroute with about 0.001 mg to about 1.0 mg, preferably about 0.1 mg, ofHuman β1A sodium channel subunit in about 0.1 ml buffer or salineincorporated in an equal volume of an acceptable adjuvant, as discussedabove. Freund's adjuvant is preferred, with Freund's complete adjuvantbeing used for the initial immunization and Freund's incomplete adjuvantused thereafter. The mice receive an initial immunization on day 0 andare rested for about 2 to about 30 weeks. Immunized mice are given oneor more booster immunizations of about 0.001 to about 1.0 mg of Humanβ1A sodium channel subunit in a buffer solution such as phosphatebuffered saline by the intravenous (IV) route.

Lymphocytes, from antibody positive mice, preferably spleniclymphocytes, are obtained by removing spleens from immunized mice bystandard procedures known in the art. Hybridoma cells are produced bymixing the splenic lymphocytes with an appropriate fusion partner,preferably myeloma cells, under conditions that will allow the formationof stable hybridomas. Fusion partners may include, but are not limitedto: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp2/0, with Sp2/0being generally preferred. The antibody producing cells and myelomacells are fused in polyethylene glycol, about 1000 mol. wt., atconcentrations from about 30% to about 50%. Fused hybridoma cells areselected by growth in hypoxanthine, thymidine and aminopterinsupplemented Dulbecco's Modified Eagles Medium (DMEM) by proceduresknown in the art. Supernatant fluids are collected from growth positivewells on about days 14, 18, and 21 and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay(SPIRA) or ELISA using Human β1A sodium channel subunit as the antigen.The culture fluids can also be tested in the Ouchterlony precipitationassay to determine the isotype of the mAb. Hybridoma cells from antibodypositive wells are cloned by a technique such as the soft agar techniqueof MacPherson, Soft Agar Techniques, in Tissue Culture Methods andApplications, Kruse and Paterson, Eds., Academic Press, 1973 or by thetechnique of limited dilution.

Monoclonal antibodies are produced in vivo by injection of pristaneprimed Balb/c mice, approximately 0.5 ml per mouse, with about 1×10⁶ toabout 6×10⁶ hybridoma cells at least about 4 days after priming. Ascitesfluid is collected at approximately 8-12 days after cell transfer andthe monoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-Human β1A sodium channel subunit mAb iscarried out by growing the hybridoma in tissue culture media well knownin the art. High density in vitro cell culture may be conducted toproduce large quantities of anti-human β1A sodium channel subunit mAbsusing hollow fiber culture techniques, air lift reactors, roller bottle,or spinner flasks culture techniques well known in the art. The mAb arepurified by techniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of Human β1Asodium channel subunit in body fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for Human β1A sodium channel subunitpolypeptide fragments, or full-length nascent Human β1A sodium channelsubunit polypeptide, or the individual Human β1A sodium channel subunitsubunits. Specifically, it is readily apparent to those skilled in theart that monospecific antibodies may be generated which are specific foronly one Human β1A sodium channel subunit or the fully functional humanβ1A sodium channel subunit protein. It is also apparent to those skilledin the art that monospecific antibodies may be generated that inhibitnormal function of human β1A sodium channel subunit protein.

Human β1A sodium channel subunit antibody affinity columns are made byadding the antibodies to a gel support such that the antibodies formcovalent linkages with the gel bead support. Preferred covalent linkagesare made through amine, aldehyde, or sulfhydryl residues contained onthe antibody. Methods to generate aldehydes or free sulfhydryl groups onantibodies are well known in the art; amine groups are reactive with,for example, N-hydroxysuccinimide esters.

Kit Compositions Containing Human β1A Sodium Channel Subunit SpecificReagents

Kits containing Human β1A sodium channel subunit DNA or RNA, antibodiesto Human β1A sodium channel subunit, or Human β1A sodium channel subunitprotein may be prepared. Such kits are used to detect DNA whichhybridizes to Human β1A sodium channel subunit DNA or to detect thepresence of Human β1A sodium channel subunit protein or peptidefragments in a sample. Such characterization is useful for a variety ofpurposes including, but not limited to, forensic analyses, diagnosticapplications, and epidemiological studies.

The DNA molecules, RNA molecules, recombinant protein and antibodies ofthe present invention may be used to screen and measure levels of Humanβ1A sodium channel subunit DNA, Human β1A sodium channel subunit RNA orHuman β1A sodium channel subunit protein. The recombinant proteins, DNAmolecules, RNA molecules and antibodies lend themselves to theformulation of kits suitable for the detection and typing of Human β1Asodium channel subunit. Such a kit would comprise a compartmentalizedcarrier suitable to hold in close confinement at least one container.The carrier would further comprise reagents such as recombinant Humanβ1A sodium channel subunit protein or anti-human β1A sodium channelsubunit antibodies suitable for detecting Human β1A sodium channelsubunit. The carrier may also contain a means for detection such aslabeled antigen or enzyme substrates or the like.

Gene Therapy

Nucleotide sequences that are complementary to the Human β1A sodiumchannel subunit encoding DNA sequence can be synthesized for antisensetherapy. These antisense molecules may be DNA, stable derivatives of DNAsuch as phosphorothioates or methylphosphonates, RNA, stable derivativesof RNA such as 2′-O-alkylRNA, or other Human β1A sodium channel subunitantisense oligonucleotide mimetics. Human β1A sodium channel subunitantisense molecules may be introduced into cells by microinjection,liposome encapsulation or by expression from vectors harboring theantisense sequence. Human β1A sodium channel subunit antisense therapymay be particularly useful for the treatment of diseases where it isbeneficial to reduce Human β1A sodium channel subunit activity.

Human β1A sodium channel subunit gene therapy may be used to introduceHuman β1A sodium channel subunit into the cells of target organisms. TheHuman β1A sodium channel subunit gene can be ligated into viral vectorsthat mediate transfer of the Human β1A sodium channel subunit DNA byinfection of recipient host cells. Suitable viral vectors includeretrovirus, adenovirus, adeno-associated virus, herpes virus, vacciniavirus, polio virus and the like. Alternatively, Human β1A sodium channelsubunit DNA can be transferred into cells for gene therapy by non-viraltechniques including receptor-mediated targeted DNA transfer usingligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofectionmembrane fusion or direct microinjection. These procedures andvariations thereof are suitable for ex vivo as well as in vivo Human β1Asodium channel subunit gene therapy. Human β1A sodium channel subunitgene therapy may be particularly useful for the treatment of diseaseswhere it is beneficial to elevate Human β1A sodium channel subunitactivity. Protocols for molecular methodology of gene therapy suitablefor use with the human β1A sodium channel subunit gene is described inGene Therapy Protocols, edited by Paul D. Robbins, Human press, TotawaN.J., 1996.

Pharmaceutical Compositions

Pharmaceutically useful compositions comprising Human β1A sodium channelsubunit DNA, Human, β1A sodium channel subunit RNA, or Human β1A sodiumchannel subunit protein, or modulators of Human β1A sodium channelsubunit receptor activity, may be formulated according to known methodssuch as by the admixture of a pharmaceutically acceptable carrier.Examples of such carriers and methods of formulation may be found inRemington's Pharmaceutical Sciences. To form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain an effective amount of the protein, DNA, RNA,or modulator.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose disorders inwhich modulation of Human β1A sodium channel subunit-related activity isindicated. The effective amount may vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration. The pharmaceuticalcompositions may be provided to the individual by a variety of routessuch as subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties that are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages defined by routine testing in order toobtain optimal inhibition of the Human β1A sodium channel subunitreceptor or its activity while minimizing any potential toxicity. Inaddition, co-administration or sequential administration of other agentsmay be desirable.

The present invention also has the objective of providing suitabletopical, oral, systemic and parenteral pharmaceutical formulations foruse in the novel methods of treatment of the present invention. Thecompositions containing compounds or modulators identified according tothis invention as the active ingredient for use in the modulation ofHuman β1A sodium channel subunit can be administered in a wide varietyof therapeutic dosage forms in conventional vehicles for administration.For example, the compounds or modulators can be administered in suchoral dosage forms as tablets, capsules (each including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, solutions, suspensions, syrups and emulsions, or byinjection. Likewise, they may also be administered in intravenous (bothbolus and infusion), intraperitoneal, subcutaneous, topical with orwithout occlusion, or intramuscular form, all using forms well known tothose of ordinary skill in the pharmaceutical arts. An effective butnon-toxic amount of the compound desired can be employed as a Human β1Asodium channel subunit modulating agent.

The daily dosage of the products may be varied over a wide range from0.01 to 1,000 mg per patient, per day. For oral administration, thecompositions are preferably provided in the form of scored or unscoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 100 mg/kg of body weight per day. The rangeis more particularly from about 0.001 mg/kg to 10 mg/kg of body weightper day. The dosages of the Human β1A sodium channel subunit receptormodulators are adjusted when combined to achieve desired effects. On theother hand, dosages of these various agents may be independentlyoptimized and combined to achieve a synergistic result wherein thepathology is reduced more than it would be if either agent were usedalone.

Advantageously, compounds or modulators of the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.Furthermore, compounds or modulators for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds or modulators of the presentinvention is selected in accordance with a variety of factors includingtype, species, age, weight, sex and medical condition of the patient;the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

In the methods of the present invention, the compounds or modulatorsherein described in detail can form the active ingredient, and aretypically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orβ-lactose, corn sweeteners, natural and synthetic gums such as acacia,tragacanth or sodium alginate, carboxymethylcellulose, polyethyleneglycol, waxes and the like. Lubricants used in these dosage formsinclude, without limitation, sodium oleate, sodium stearate, magnesiumstearate, sodium benzoate, sodium acetate, sodium chloride and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents that may be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations, which generally containsuitable preservatives, are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eolis, mineral oil, PPG2 myristyl propionate, and the like, to form,e.g., alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions, and shampoos in cream or gel formulations.

The compounds or modulators of the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds or modulators of the presentinvention may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinyl-pyrrolidone, pyrancopolymer, polyhydroxypropylmethacrylamidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds ormodulators of the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels.

For oral administration, the compounds or modulators may be administeredin capsule, tablet, or bolus form or alternatively they can be mixed inthe animals feed. The capsules, tablets, and boluses are comprised ofthe active ingredient in combination with an appropriate carrier vehiclesuch as starch, talc, magnesium stearate, or di-calcium phosphate. Theseunit dosage forms are prepared by intimately mixing the activeingredient with suitable finely-powdered inert ingredients includingdiluents, fillers, disintegrating agents, and/or binders such that auniform mixture is obtained. An inert ingredient is one that will notreact with the compounds or modulators and which is non-toxic to theanimal being treated. Suitable inert ingredients include starch,lactose, talc, magnesium stearate, vegetable gums and oils, and thelike. These formulations may contain a widely variable amount of theactive and inactive ingredients depending on numerous factors such asthe size and type of the animal species to be treated and the type andseverity of the infection. The active ingredient may also beadministered as an additive to the feed by simply mixing the compoundwith the feedstuff or by applying the compound to the surface of thefeed. Alternatively the active ingredient may be mixed with an inertcarrier and the resulting composition may then either be mixed with thefeed or fed directly to the animal. Suitable inert carriers include cornmeal, citrus meal, fermentation residues, soya grits, dried grains andthe like. The active ingredients are intimately mixed with these inertcarriers by grinding, stirring, milling, or tumbling such that the finalcomposition contains from 0.001 to 5% by weight of the activeingredient.

The compounds or modulators may alternatively be administeredparenterally via injection of a formulation consisting of the activeingredient dissolved in an inert liquid carrier. Injection may be eitherintramuscular, intraluminal, intratracheal, or subcutaneous. Theinjectable formulation consists of the active ingredient mixed with anappropriate inert liquid carrier. Acceptable liquid carriers include thevegetable oils such as peanut oil, cotton seed oil, sesame oil and thelike as well as organic solvents such as solketal, glycerol formal andthe like. As an alternative, aqueous parenteral formulations may also beused. The vegetable oils are the preferred liquid carriers. Theformulations are prepared by dissolving or suspending the activeingredient in the liquid carrier such that the final formulationcontains from 0.005 to 10% by weight of the active ingredient.

Topical application of the compounds or modulators is possible throughthe use of a liquid drench or a shampoo containing the instant compoundsor modulators as an aqueous solution or suspension. These formulationsgenerally contain a suspending agent such as bentonite and normally willalso contain an antifoaming agent. Formulations containing from 0.005 to10% by weight of the active ingredient are acceptable. Preferredformulations are those containing from 0.01 to 5% by weight of theinstant compounds or modulators.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1 Cloning of Human Sodium Channel β1A Subunit

Rapid Amplification of cDNA End (RACE-PCR): Marathon-Ready™ humanadrenal gland cDNA library (Cat. No. 7430-1) was purchased from Clontech(Palo Alto, Calif.). Primary Race PCR was performed in 50 μl finalvolume. The reaction mixture contains 5 μl of Marathon-Ready™ humanadrenal gland cDNA, 5 μl of 10× reaction buffer, 200 μM dNTP, 200 nM AP1primer (Clontech, 5′-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3′SEQ.ID.NO.:01), 200 nM human sodium channel β1 subunit specific primerSB1-10 (5′-TGG ACC TTC CGC CAG AAG GGC ACTG-3′ SEQ.ID.NO.:02) and 1 μlof 50× Advatage2 DNA polymerase mixture (Clontech). The thermal cyclerparameter for RACE-PCR was: initial denaturing at 94° C. for 30 sec, 5cycles of 94° C./5 sec and 72° C./4 min, 5 cycles of 94° C./5 sec and70° C./4 min, and 20 cycles of 94° C./5 sec and 68° C./4 min.

Assessment of RACE-PCR product: The quality of the RACE-PCR reaction wasassessed by testing whether the carboxyl terminus of human sodiumchannel β₁ subunit had been amplified. This was done by PCR with 1 μl ofRACE-PCR product as template, and 200 nM of two human sodium channel β₁subunit specific primers. The specific primers are: a forward primerSB1-11 (5′-CTG GAG GAG GAT GAG CGC TTC GAG-3′ SEQ.ID.NO.:3) located atdownstream of SB 1-10 and a reverse primer SB 1-13 (5′-CTA TTC GGC CACCTG GAC GCC-3′ SEQ.ID.NO.:4) located at the end of human sodium channelβ₁ subunit. The PCR reaction parameter was: initial denaturing at 94° C.for 1 min, followed by 30 cycles of denaturing at 94° C. for 20 sec,annealing at 55° C. for 20 sec and extension at 72° C. for 1 min. Aspredicted, a strong 0.5-kb DNA fragment was observed, suggesting theRACE-PCR was successful in term of amplification of carboxyl terminus ofhuman sodium channel β₁ subunit.

Cloning of RACE-PCR Products into pPCR-Script vector: The RACE-PCRproduct was cloned with PCR-Script™ Amp Cloning Kit (Statagene, Calif.)following the company provided protocol. Briefly, the RACE-PCR productwas first purified by StrataPrep PCR purification column. Ten μl ofpurified RACE-PCR product was then blunt-ended at 72° C. for 30 min infinal volume of 13 μl with 10 μl of purified RACE-PCR product, 1.3 μl ofpolishing buffer, 1 μl of 10 mM dNTP and 1 μl of 0.5 U/μl cloned Pfu DNApolymerase. The ligation was performed at room temperature for 1 hour ina final volume of 10 ml containing 4.5 μl of blunt-ended RACE-PCRproduct, 1 μl of 10 ng/μl pPCR-Script cloning vector, 1 μl of PCR-Script10× reaction buffer, 0.5 μl of 10 mM rATP, 11 of 5 U/μl of SrfIrestriction enzyme and 1 μl of 4 U/μl of T4 DNA ligase. The ligationreaction was stopped by heating the sample at 65° C. for 10 min, andfinally, 2 μl of ligation mixture was transformed into XL10-Goldbacteria.

Identification of Human Sodium Channel β_(1A) Clone: Since human sodiumchannel β₁ subunit specific primer was used for RACE-PCR of carboxylterminus of β_(1A) subunit, theoretically, both β₁ and β_(1A) subunitswill be cloned. To exclude β₁ subunit, individual clones werecharacterized by PCR with a pair of primers for specific amplificationof β₁ subunit. The primers used in the PCR are: forward primer SB1-17(5′-GTG TCT GAG ATC ATG ATG-3′ SEQ.ID.NO.:5) and reverse primer SB1-13(see above). The PCR reaction parameter was: initial denaturing at 94°C. for 1 min, followed by 30 cycles of denaturing at 94° C. for 20 sec,annealing at 55° C. for 20 sec and extension at 72° C. for 1 min. Allthe PCR negative clones are non-β₁ subunit and subjected to furthersequencing analysis.

Cloning of sodium channel β_(1A) subunit for human fetal brain cDNA:With human sodium channel β_(1A) subunit specific primers obtained byRACE-PCR, sodium channel β_(1A) was also cloned from Marathon-Ready™human fetal brain cDNA library (Clontech cat. No. 7402-1, Palo Alto,Calif.). PCR was performed in 50 μl final volume, containing 5 μl ofMarathon-Ready™ human adrenal gland cDNA, 5 μl of 10× reaction buffer,200 μM dNTP, 200 nM SB1-6 primer (5′-GCC ATG GGG AGG CTG CTG GCC TTA GTGGTC-3′ SEQ.ID.NO.:6) and SB1-19 primer (5′-GTG TGC CTG CAG CTG CTC AA-3′SEQ.ID.NO.:7) and 1 μl of 50× Advantage2 DNA polymerase mixture(Clontech). The PCR reaction parameter was: initial denaturing at 94° C.for 1 min, followed by 30 cycles of denaturing at 94° C. for 20 sec,annealing at 55° C. for 20 sec and extension at 72° C. for 1 min. Fourindependent clones were picked and subjected to double strained DNAsequencing analysis. All of the four independent clones from human fetalbrain have identical sequences to RACE-PCR cloned β_(1A) subunit fromthe human adrenal gland. The coding region of the human β1A nucleic acidis provided as SEQ ID NO: 12 and the entire isolated cDNA, includinguntranslated regions (UTR) is provided as SEQ ID NO:13. The5′-untranslated region is the same as human voltage gated sodium channelβ1 subunit. The 3′ untranslated region is about 160 bp in length withoutthe polyA tract. Translation begins at the ATG beginning at nucleotide 4and ends at the stop codon at nucleotide 808.

EXAMPLE 2

Primary Structure of the Human Voltage Gated Sodium Channel β1A SubunitProtein and Genomic Structure of β1 and β1A Gene (SCN1B)

By using a RACE-PCR technique, a novel human sodium channel β1A subunitwas cloned. The cDNA contained an 807-base pair coding region for 268amino acids and a 164-base pair 3′-untranslated region. The translatedamino acid sequence is provided as SEQ ID NO:14 and includes a signalsequence and potential N-linked glycosylation sites as well as atransmembrane domain. The clone, designated β1A, is related to thesodium channel β1 subunit. The conserved motifs found in the sodiumchannel β1 subunit family include a signal peptide sequence,extracellular immunoglobulin fold domain and carboxyl terminaltransmembrane domain. The predicted peptide contains hydrophobicamino-terminal residues (1-16 residues) with sequences highly predictiveof the signal cleavage sites that would result in mature proteinsinitiating at amino acid 17 [Alanine]. The hydrophobic carboxyl terminalresidues (243-262 residues) may serve as a transmembrane domain. Theestimated protein molecular mass is about 28.8 kD after removing thesignal peptide from the amino terminus. The cloned human sodium channelβ1A subunit migrated with an apparent Mr of 32 kDa when analyzed by8-20% SDS/PAGE. Peptide sequence comparison reveals that the predictedpeptide is 72% identical to both the human sodium channel 11 subunit andthe rat VGSC β1A subunit. Like the rat sodium channel β1A subunit, thehuman sodium channel β1A subunit contains an amino terminal region(1-149) of 100% identity to human 01 subunit and a novel carboxylterminal region (150-268) with a less than 17% identity to that of humanP1 subunit (FIG. 1). The genomic organization study of the human sodiumchannel β1 subunit gene, SCN1B (Makita, et al. 1994) has revealed thatthe gene spans about 9 kb with six exons and five introns on chromosome19 (19q13.1-q13.2). Blast searching of human genomic sequences revealedthat the amino terminal region of human sodium channel β1A subunit(1-149) are encoded by exon 1-3, while the novel carboxyl terminalregion was encoded by intron 3 adjacent to exon 3 (FIG. 2). Since thesite of divergence between the β1 and β1A subunits cDNA was locatedprecisely at the exon 3-intron 3 boundary of the SCN1B gene, the humansodium channel β1A subunit should be considered as a splicing variant ofthe β1 subunit via the retained intron with an in-frame stop codon.However, the carboxyl terminal region of the human VGSC β1A subunit isless than 33% identical to the rat VGSC β1A subunit. Blast searching ofhuman genomic sequence with the cDNA encoding rat β1A carboxyl terminusfailed to identify any homologous region in the human β1 gene, whichstrongly suggests that both rat and human β1A carboxyl termini areencoded by intron 3 and the difference is due to the speciesdifferences.

Blast searching of the NIH database with the carboxyl terminal sequenceof the human sodium channel β1A subunit also revealed an unassignedclone (Accession number: AI742310) in the human EST database, which wascloned from the pool of five normalized cDNA libraries. The cloneAI742310 was shorter than, but 100% identical to the carboxyl terminalregion (150-268 residues) of the human sodium channel β_(1A). Thesimilar blast searching failed to reveal any sequence with more than 25%identity from any other databases.

EXAMPLE 3 Generation of Polyclonal Antibodies

Two peptide sequences (HB1A-1 and HB1A-2) derived from carboxyl terminusof human β1A subunit were selected for raising polyclonal antibodies inrabbits. The amino acid sequences are: (HB1A-1) AC-RWRDRWQAVDRTGC-AMIDE(SEQ IN NO.:08) and (HB1A-2) AC-CVPHRRSGYRTQL-AMIDE (SEQ IN NO.:09). Thepeptides were synthesized and antibodies were raised and purified byBioSource International, Inc. The antibodies were tested by ELISAagainst the antigen peptides and affinity purified with the samepeptides. Serum and affinity purified antibodies were used forimmunoanalysis, such as Western blot, immunoprecipitation,immunocytochemistry and immunohistochemistry.

EXAMPLE 4

In Vitro Translation Analysis of Human Sodium Channel β1A Subunit.

The cDNA of the human sodium channel β1A subunit was first subclonedinto pAGA3 vector, which was engineered for high efficiency of in vitrotranscription and translation. Briefly, the cDNA fragment encoding humansodium channel β1A subunit was excised from the pcDNA3.1 construct (SeeExample 6) by digested with NcoI and XbaI. An about 900 bp cDNA fragmentwas separated by 1% agarose gel and purified by Qiaquick SpinPurification Kit (Qiagen). The vector pAGA3 was also digested with NcoIand XbaI restriction enzymes. The purified liner vector was ligated withthe cDNA fragment and transformed into bacteria. The recombinants wasisolated and confirmed by restriction enzyme digestion and DNAsequencing. In vitro translation of the human sodium channel β1A subunitwas done with TnT® T7 Quick Coupled Transcription/Translation System(Promega) following the vendor recommended protocol. Briefly, 1 μghβ1A/pAGA3 construct was added to 40 μl of TNT Quick Master Mix with 2μl of [³⁵S]-methionine (1000 Ci/mmmol at 10 mCi/ml) in a final volume of50 μl. The reaction mixture was incubated at 30° C. for 90 min. Five μlof reaction mixture was mixed with an equal volume of SDS/PAGE loadingbuffer and subjected to 8-20% SDS/PAGE for analysis. Afterelectrophoresis, the gel was stained with Coomassie Blue R250, dried andexposed to X-ray film. Both the human β1 and human β1A migrate to themolecular weight predicted by translation of the amino acid sequencesfrom the corresponding nucleic acid sequences.

The in vitro translated human sodium channel β1A subunit was alsoanalyzed by Western blot and immunoprecipitation.

EXAMPLE 5

Northern Blot Analysis of the Human β1A Sodium Channel SubunitExpression

A northern blot was used to analyze the tissue distribution of the humansodium channel β1A subunit. The cDNA fragment encoding 217-268 residuesof the human sodium channel β1A was used as a probe. To make the probe,a 153 bp DNA fragment was first amplified from the DNA construct(NQC130) containing full length cDNA of human sodium channel β1A withhuman sodium channel β1A specific primers SB1-25 (5′-T CAA AGC ATG CCTGTC CC-3′ SEQ.ID.NO.:10) and SB 1-20 (5′-TCA AAC CAC ACC CCG AGA AA-3′SEQ.ID.NO.:11). Following amplification, the PCR product was directlycloned into the pCR2.1 vector (Invitrogen) and was further confirmed byDNA sequencing. The resulting construct NQC226 was digested with therestriction enzyme HindIII and DNA was then precipitated after phenoland chloroform extraction. To label the probe, the antisense cDNAfragment was linearly amplified with Strip-EZ™ PCR kit (Ambion Tex.).The reaction was performed in 20 μl final volume, containing 25 nglinearized NQC226, 2 μl of 10× reaction buffer, 2 μl of 10× dNTPsolution, 2 μl of 3000 Ci/mmol [α-³²P]-dATP (Amersham PharmaciaBiotech), 1 U thermostable DNA polymerase and 2 μl of 10 pmo/μl ofSB1-20 primer. The PCR reaction parameter was: initial denaturing at 94°C. for, 1 min, followed by 35 cycles of denaturing at 94° C. for 20 sec,annealing at 55° C. for 20 sec and extension at 72° C. for 1 min. Thelabeled probe was then separated from free [α-³²P] dATP with MicroSpin™G-50 column (Amersham Pharmacia Biotech).

Human MTN™ (Multiple Tissue Northern) blot (Cal. No. 7760-1) and HumanBrain MTN™ Blot II (7755-1) were purchased from Clontech (Palo Alto,Calif.). The blots were pre-hybridized with 5 ml UltraHyb Solution(Ambion, 1×) at 42° C. for 2 hours, and then hybridized in the presenceof 1×10⁶ cpm/ml probe of human sodium channel β1A subunit at 42° C.overnight. The blots were washed with 2×200 ml of 0.2×SSC/0.1% SDSsolution at 65° C. for two hours. Finally the blots were exposed toX-ray film in −80° C. freezer overnight.

2.0 kb cDNA fragment encoding human β-actin was used as the controlprobe. The same blots were stripped at 68° C. for 15 minutes withStrip-EZ™ removal kit provided by Ambion. The blots were thenpre-hybridized with 5 ml of UltraHyb at 42° C. for 2 hour, and thenhybridized in the presence of human β-actin probe for 2 hours at 42° C.The blots were washed with 2×200 ml of 0.2×SSC/0.1% SDS solution at 68°C. for two hours. Finally the blots were exposed to X-ray film in −80°C. freezer for 1 hour.

Northern blot analysis demonstrated that the VGSC β1A subunit was highlyexpressed in, but not limited to, most regions of human brain andskeletal muscle. Lower levels of expression were observed in heart,placenta, liver, kidney and pancrease. In brain, the VGSC β1A subunitwas expressed most highly in the cerebellum region. The size of the VGSCβ1A subunit transcripts varied slightly in different tissues. However,the major transcript was about 7.5 kb in size.

EXAMPLE 6

Cloning of Human β1A Sodium Channel Subunit cDNA into a MammalianExpression Vector

The cDNA of human sodium channel β1A subunit were cloned into themammalian expression vectors pIRESneo (Clontech) and pcDNA3(Invitrogen). The cDNA fragments encoding the human sodium channel β1Asubunit were excised from pPCR-Script plasmids (NQC128) by digestionwith BamHI and Not I, separated by agarose gel electrophoresis andpurified by Qiaquick Spin purification kit (Qiagen). The vector pIRESneowas also linearized with BamHI and NotI and purified, and ligated withcDNA fragment of hb1A isolated from NQC128. Recombinants were isolated,and confirmed by restriction enzyme digestion and DNA sequencing. Theclones NQC141 (hβ1A/pIRESneo) were then used to transfect humanneuroblastoma cells (SK-N-SH) by SuperFect (Qiagen) following thevendor's protocol. Stable cell clones are selected by growth in thepresence of G418. Single G418 resistant clones are isolated and shown tocontain the intact Human β1A sodium channel subunit gene. Clonescontaining the Human β1A sodium channel subunit cDNAs are analyzed forexpression using immunological techniques, such as Western blot,immunoprecipitation, and immunofluorescence using antibodies specific tothe Human β1A sodium channel subunit proteins. Antibody is obtained fromrabbits inoculated with peptides that are synthesized from the aminoacid sequence predicted from the Human β1A sodium channel subunitsequences. Expression is also analyzed using sodium influx assay.

The Human β1A sodium channel subunit gene was inserted into pcDNA3.1(Invitrogen). The cDNA fragment encoding human sodium channel β1Asubunit was excised from NQC130 plasmid by digested with XhoI and NotI,and the cDNA inserts isolated by agarose gel electrophoresis andpurified by Qiaquick Spin Purification Kit (Qiagen). The vector, pcDNA3,was digested with BamHI and NotI, and the linear vector isolated by gelelectrophoresis, and ligated with cDNA inserts. Recombinant plasmidscontaining human sodium channel β1A subunit were isolated, and confirmedby restriction enzyme digestion and DNA sequencing. The clone NQC139(hβ1A/pcDNA3.1) was used to transiently transfect human neuroblastomacells (SK-N-SH) by SuperFect (Qiagen) and the function of hb1A onmodulation of sodium influx was tested by sodium influx assay.

Cells that are expressing Human sodium channel β1A subunit, stably ortransiently, are used to test for expression of channel and for ligandbinding activity. These cells are used to identify and examine othercompounds for their ability to modulate, inhibit or activate the channeland to compete for radioactive ligand binding.

Cassettes containing the human sodium channel β1A subunit cDNA in thepositive orientation with respect to the promoter are ligated intoappropriate restriction sites 3′ of the promoter and identified byrestriction site mapping and/or sequencing. These cDNA expressionvectors are introduced into fibroblastic host cells for example COS-7(ATCC# CRL1651), and CV-1 tat [Sackevitz et al., Science 238: 1575(1987)], 293, L (ATCC# CRL6362)] by standard methods including but notlimited to electroporation, or chemical procedures (cationic liposomes,DEAE dextran, calcium phosphate). Transfected cells and cell culturesupernatants can be harvested and analyzed for human sodium channel β1Asubunit expression as described herein.

All of the vectors used for mammalian transient expression can be usedto establish stable cell lines expressing Human β1A sodium channelsubunit. Unaltered Human β1A sodium channel subunit cDNA constructscloned into expression vectors are expected to program host cells tomake Human β1A sodium channel subunit protein. In addition, Human β1Asodium channel subunit is expressed extracellularly as a secretedprotein by ligating Human β1A sodium channel subunit cDNA constructs toDNA encoding the signal sequence of a secreted protein. The transfectionhost cells include, but are not limited to, CV-1-P [Sackevitz et al.,Science 238: 1575 (1987)], tk-L [Wigler, et al. Cell 11: 223 (1977)],NS/0, and dHFr-CHO [Kaufman and Sharp, J. Mol. Biol. 159: 601, (1982)].

Co-transfection of any vector containing Human β1A sodium channelsubunit cDNA with a drug selection plasmid including, but not limited toG418, aminoglycoside phosphotransferase; hygromycin, hygromycin-Bphosphotransferase; APRT, xanthine-guanine phosphoribosyl-transferase,will allow for the selection of stably transfected clones. Levels ofHuman β1A sodium channel subunit are quantitated by the assays describedherein.

Human β1A sodium channel subunit cDNA constructs are also ligated intovectors containing amplifiable drug-resistance markers for theproduction of mammalian cell clones synthesizing the highest possiblelevels of Human β1A sodium channel subunit. Following introduction ofthese constructs into cells, clones containing the plasmid are selectedwith the appropriate agent, and isolation of an over-expressing clonewith a high copy number of plasmids is accomplished by selection inincreasing doses of the agent.

The expression of recombinant Human β1A sodium channel subunit isachieved by transfection of full-length Human β1A sodium channel subunitcDNA into a mammalian host cell.

EXAMPLE 7

Cloning of Human β1A Sodium Channel Subunit cDNA into a BaculovirusExpression Vector for Expression in Insect Cells

Baculovirus vectors, which are derived from the genome of the AcNPVvirus, are designed to provide high level expression of cDNA in the Sf9line of insect cells (ATCC CRL# 1711). Recombinant baculovirusesexpressing Human β1A sodium channel subunit cDNA is produced by thefollowing standard methods (InVitrogen Maxbac Manual): the Human β1Asodium channel subunit cDNA constructs are ligated into the polyhedringene in a variety of baculovirus transfer vectors, including the pAC360and the BlueBac vector (InVitrogen). Recombinant baculoviruses aregenerated by homologous recombination following co-transfection of thebaculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res. 18: 5667 (1990)] into Sf9 cells. Recombinant pAC360viruses are identified by the absence of inclusion bodies in infectedcells and recombinant pBlueBac viruses are identified on the basis ofβ-galactosidase expression (Summers, M. D. and Smith, G. E., TexasAgriculture Exp. Station Bulletin No. 1555). Following plaquepurification, Human β1A sodium channel subunit expression is measured bythe assays described herein.

The cDNA encoding the entire open reading frame for the Human β1A sodiumchannel subunit is inserted into the BamHI site of pBlueBacII.Constructs in the positive orientation are identified by sequenceanalysis and used to transfect Sf9 cells in the presence of linear AcNPVmild type DNA.

Authentic, active Human β1A sodium channel subunit is found in thecytoplasm of infected cells. Active Human β1A sodium channel subunit isextracted from infected cells by hypotonic or detergent lysis.

EXAMPLE 8

Cloning of Human β1A Sodium Channel Subunit cDNA into a Yeast ExpressionVector

Recombinant Human β1A sodium channel subunit is produced in the yeast S.cerevisiae following the insertion of the optimal Human β1A sodiumchannel subunit cDNA cistron into expression vectors designed to directthe intracellular or extracellular expression of heterologous proteins.In the case of intracellular expression, vectors such as EmBLyex4 or thelike are ligated to the Human β1A sodium channel subunit cistron [Rinas,U. et al., Biotechnology 8: 543-545 (1990); Horowitz B. et al., J. Biol.Chem. 265: 4189-4192 (1989)]. For extracellular expression, the Humanβ1A sodium channel subunit cistron is ligated into yeast expressionvectors which fuse a secretion signal (a yeast or mammalian peptide) tothe NH₂ terminus of the Human β1A sodium channel subunit protein[Jacobson, M. A., Gene 85: 511-516 (1989); Riett L. and Bellon N.Biochem. 28: 2941-2949 (1989)].

These vectors include, but are not limited to pAVE1>6, which fuses thehuman serum albumin signal to the expressed cDNA [Steep O. Biotechnology8: 42-46 (1990)], and the vector pL8PL which fuses the human lysozymesignal to the expressed cDNA [Yamamoto, Y., Biochem. 28: 2728-2732)]. Inaddition, Human β1A sodium channel subunit is expressed in yeast as afusion protein conjugated to ubiquitin utilizing the vector pVEP [Ecker,D. J., J. Biol. Chem. 264: 7715-7719 (1989), Sabin, E. A., Biotechnology7: 705-709 (1989), McDonnell D. P., Mol. Cell Biol. 9: 5517-5523(1989)]. The levels of expressed Human β1A sodium channel subunit aredetermined by the assays described herein.

EXAMPLE 9

Purification of Recombinant Human β1A Sodium Channel Subunit

Recombinantly produced Human β1A sodium channel subunit may be purifiedby antibody affinity chromatography.

Human β1A sodium channel subunit antibody affinity columns are made byadding the anti-human β1A sodium channel subunit antibodies toAffigel-10 (Biorad), a gel support which is pre-activated withN-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1 M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23 M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) together withappropriate membrane solubilizing agents such as detergents and the cellculture supernatants or cell extracts containing solubilized Human β1Asodium channel subunit are slowly passed through the column. The columnis then washed with phosphate-buffered saline together with detergentsuntil the optical density (A280) falls to background, then the proteinis eluted with 0.23 M glycine-HCl (pH 2.6) together with detergents. Thepurified Human β1A sodium channel subunit protein is then dialyzedagainst phosphate buffered saline.

EXAMPLE 10 Immunohistochemistry of Human Tissue

Immunohistochemistry: Protocols for immunohistochemistry have beenpreviously described (D'Andrea, et al, 1998). All incubations wereperformed at room temperature. After microwaving the slides in Target(Dako, Carpenturia, Calif.), the slides were placed in PBS, then 3%H₂O₂, rinsed in PBS and then appropriate blocking serum was added for 10minutes. Subsequently, primary antibody, either rabbit polyclonalanti-human hβ1A antibody (HB1A-1, see Example 3) at a titer of 1:200 orrabbit polyclonal anti-rat β1A antibody at a titer of 1:400 was appliedto the slides for 30 minutes. Proper species isotype antibody (VectorLabs, Burlingham, Calif.) was substituted as the primary antibody forthe negative control. After several PBS washes, a biotinylated secondaryantibody (Vector Labs) was placed on the slides for 30 minutes.Subsequently, the slides were washed in PBS and then the avidin-biotincomplex (ABC, Vector Labs) was applied to the cells for 30 minutes. Thepresence of the primary antibodies was detected by adding DAB(3′-diaminobenzidine HCl; Biomeda, Foster City, Calif.) for 2 times 5minutes. Slides were briefly exposed to Mayer's hematoxylin for 1minute, dehydrated and coverslipped.

Results:

With anti-human β1A antibody: We were able to detect intracellular β1Ain the human DRG neurons. We also localized membrane-associated β1A inthe neuronal fibers of the DRG as well. We also screened a myriad ofhuman tissues and determine the β1A protein to be present in theepithelial cells of the gut (brush boarder), of the collecting tubules(distal>proximal) of the kidney (demonstrating prominent labeling), andprostate. We also observed β1A immunolabeling in the brain (cortexpyramidal neurons, cerebellar Purkinje cells, and many of the neuronalfibers throughout the brain), in the endothelial cells of the lung andother tissues, membrane of macrophages in the lung and uterus, and inthe cardiocytes of the heart. We did not observe significant, detectablelevels of β1A in the following tissues: thyroid, spleen, liver, andpancreas. We also observed similar distribution of β1A in rat tissuewith this anti-human b1a antibody.

EXAMPLE 111

Patch Clamp Analysis of VGSC β1A Subunit Expressed in Xenopus Oocytes

1. In vitro synthesis of cRNA: The expression constructs of the β1A andNa_(V)1.2 (a type II alpha subunit of the voltage gated calcium channelprotein family obtained from Dr. A. Correa, UCLA Medical Center,Department of Anesthesiology) were linearized with restriction enzymes.The cRNAs were synthesized in vitro with T7 RNA polymerase usingreagents and protocols of the mMESSAGE mMACHINE™ transcription kit(Ambion, Austin, Tex.), with the exception that the LiCl precipitationstep was repeated twice. The cRNAs were suspended indiethylpyrocarbonate (DEPC)-treated H₂O at a final concentration of 1-2mg/ml.

2. Oocyte Isolation: Frogs (Xenopus laevis) were anesthetized byimmersion into 0.15-0.17% tricaine in water and removed from thetricaine bath. Ovarian lobes were then exposed through a small incisionmade into their abdominal wall, removed and placed into sterilizedCa²⁺-free OR-2 solution (Ca²⁺-free OR-2: 82.5 mM NaCl, 2.5 mM KCl, 1 mMMgCl₂ and 5 mM HEPES, pH adjusted to 7.6 with NaOH) and the frogsreturned to tricaine-free water for recovery. The ovarian lobes werethen rinsed with sterile water, teased open, and incubated at roomtemperature in Ca²⁺-free OR-2 containing 2 mg/ml collagenase (type I,BRL) to cause release and defolliculation of oocytes. After 1 hr on anorbital shaker (ca 60 cycles per min) the oocytes were transferred to aPetri dish with OR-2. Dead and too small oocytes were removed byaspiration and the selected oocytes were washed several times withcollagenase free and Ca²⁺-free OR-2 solution, incubated under agitationfor an additional hour with solution changes every 7-8 min, and placedinto an incubator at 19° C. and incubated for an additional 1 hr in a1:4 mixture of sterile SOS and Ca²⁺-free OR-2 solutions (SOS: 100 mMNaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, pH adjusted to 7.6with NaOH) and 30 min in 1:3 SOS/Ca²⁺-free OR-2. The oocytes were thenplaced into 100% SOS, sorted once more and kept at 19° C.

3. Injections: Oocytes were injected with 50 nl containing the cRNAs tobe translated at a final concentration of 100 μg/ml of each species, andoligonucleotides at the indicated concentrations. Injections wereperformed immediately after isolation or at varying times thereafter forup to three days. Injected oocytes were kept at 17-19° C. in sterile SOScontaining 50 μg/ml gentamycin with daily solution changes until usedfor electrophysiological testing (4-6 days post-injection).

4. Electrophysiological recordings from oocytes: The cut-open vaselinegap voltage clamp of oocyte (Taglialatela et al., 1992) was performedwith a CA-1 cut open oocyte clamp setup (Dagan, Minneapolis, Minn.). Theexperimental external solution contained 120 mM NMG-Mes, 10 mM HEPES,and 2 mM Ca(Mes)₂ at pH 7.4. The experimental internal solutioncontained 120 mM NMG-Mes, 10 mM HEPES, and 2 mM EGTA at pH 7.4. Leakageand linear capacity currents were compensated and subtracted on-lineusing p/−4 protocol from −90 mV holding potential (SHP).

Signals were filtered with an eight pole Bessel filter to ⅕ of thesampling frequency. All the recordings were performed at roomtemperature (22-23° C.).

5. Data Acquisition and Analysis: Gating and ionic currents wereacquired with a PC44 board (Innovative Technologies, Moorpark, Calif.),which interfaces with a Pentium-based computer via an IBM-compatible ATslot.

The peak G-V curve was obtained with an internal solution of 15 mM NaMesand an external solution of 1 mM NaMes (with N-methyl-glucamine (NMG) toreplace the remaining cations). The reversal potential was determinedfrom an instantaneous I-V after subtracting the gating component, andpeak conductance was calculated by dividing peak current by the voltagedifference from the reversal potential. Within the time resolution ofthe clamp, the instantaneous I-V curve was linear, so no furthercorrections were made.

6. Results: As shown in FIG. 3, the ionic current conducted by Na_(V)1.2was increased significantly by coexpression with the β1A subunit inoocytes. At the cRNA ratio of 1:20 (Na_(V)1.2 vs β1A), the ionic currentwas increased about 3 fold. The results indicate that the β1A subunitplays an important role in increasing the number of the functionalsodium channels present on the cell surface.

EXAMPLE 12

Up-Regulation and Redistribution of β1A Subunit in Rat Dorsal RootGanglia Neurons Following Spinal Nerve Ligation

1. Spinal Nerve Ligation. All procedures involving the use of animalswere carried out in accordance with the guidelines of the InstitutionalAnimal Care and Use Committee, The R.W. Johnson Pharmaceutical ResearchInstitute and American Association for Laboratory Animal Care. Spinalnerve ligation (SNL) was performed as described by Kim and Chung (1992).Briefly, male Sprague-Dawley rats (Harlan, Indianapolis, Ind.) weighingapproximately 200 g were anesthetized with isoflurane. The spinal nerveat the level of L5 or L6 was exposed through an incision left of thedorsal midline and, following removal of the left L6 transversevertebral process, tightly ligated with 6-0 silk. The incision was thenclosed with 4.0 vicryl (fascia) and skin clips. Animals were allowed torecover from anesthesia and were assessed for hind limb motor functionprior to being returned to group housing. Naive animals did not undergosurgery.

2. Evaluation of Mechanical Allodynia. Allodynia was assessed by placingrats individually on a wire mesh platform such that the plantar surfaceof each hind paw could be stimulated from below. An ascending series oftactile stimuli was applied using calibrated von Frey filaments (0.25-15g) until a rapid paw lift response was observed. An “up-down” method(Chaplan et al., 1994) was employed to determine the response thresholdin grams. Animals from which tissue was obtained were tested at severaltime points post-surgically to assure that response thresholds of theipsilateral paw were at or below the 4 g criterion. Additionally, aseparate age matched group of animals (n=3-12 ligated, n=7 naive) wastested for mechanical allodynia at various times (3 d, 10 d, 21 d, and 8wk) following surgery to profile the time course of ipsilateral andcontralateral response thresholds, as well as the response of naiveanimals.

3. Tissue Preparation. At 2 d, 14 d and 8 wk after surgery, animals(n=3-5) were sacrificed by CO₂ asphyxiation and transcardially perfusedwith 4% paraformaldehyde. The L4 and L5 DRGs were removed, processed forparaffin embedding in a multi-tissue format, serially sectioned at 5 μmand mounted onto Superfrost-Plus slides (Fisher, Pittsburgh, Pa.).

4. Immunohistochemistry. Protocols for routine immunohistochemistry(IHC) have been previously described (D'Andrea et al., 1998). Briefly,paraffin slides were dewaxed, hydrated and microwaved in Target buffer(Dako, Carpenteria, Calif.). Sections were cooled, placed inphosphate-buffered saline (PBS, pH 7.4) and then treated with 3.0% H₂O₂for 10 min to inactivate endogenous peroxidases. All subsequent reagentincubations and washes were performed at room temperature. Normalblocking serum (Vector Labs, Burlingame, Calif.) was placed on thetissues for 10 min followed by brief rinsing in PBS. Tissues were thenincubated with primary antibody for 30 min, and washed, and then treatedwith biotinylated secondary antibodies, goat anti-rabbit (Vector Labs)for 30 min. Previously characterized (Kazen-Gillespie et al., 2000)rabbit polyclonal anti-β1A and anti-β1 antibodies were used at titers of1:400. Following a rinse in PBS, tissues were treated with theavidin-biotin-HRP complex reagent (Vector Labs) for 30 min. Slides weretreated 2×5 minutes with 3,3′-diaminobenzidine (DAB, Biomeda, FosterCity, Calif.), rinsed, counterstained with hematoxylin, dehydrated,cleared in xylene and coverslipped. Monoclonal antibodies specific toneuron-specific nuclear protein (NeuN, 1:1,000; Chemicon, Temecula,Calif.) were used as a positive control to confirm tissue antigenicityand reagent quality. Negative controls included replacement of theprimary antibody with species IgG isotype non-immunized serum (VectorLabs).

5. Evaluation of β1A and β1 immunolabeling. Tissues mounted on slides ina multi-tissue format were stained simultaneously to minimize potentialstaining variability. Ipsilateral and contralateral DRG neurons withprominent nuclei from the L4 and L5 from each animal were characterizedas nociceptive if the diameter was <25 μm in diameter; all others weredesignated as sensory neurons (Oh et al., 1996; Gould et al., 1998). Thestaining of 30-40 neurons per DRG per animal was scored according to thefollowing criteria: 1) no immunoreactivity was scored as 0.0; 2) weakimmunoreactivity was scored as 1.0; 3) moderate immunoreactivity wasscored as 2.0, and 4) intense immunoreactivity was scored as 3.0. If theimmunolabeling intensity was between these whole numbered units, a 0.5increment was used (i.e. 1.5 weak to moderate labeling). These data werethen averaged per animal and per group (n=3-5 per group).

The morphology of the β1A and β1 immunoreactivity in the L5 nociceptiveand sensory DRG neurons were also characterized as 1) homogeneous: adiffuse labeling pattern; 2) punctate: several clumpy, intracellular,Nissl-like aggregates of staining; and 3) membrane: prominent peripherallabeling located predominantly along the cell membrane. Data arepresented as a percentage of each labeling pattern observed per DRG pergroup (Table 2).

Data and Statistical Analysis. The β1A and β1 staining data were groupedto identify the spinal level of the DRG (L4 or L5), laterality(contralateral or ipsilateral), neuronal type (small or large), andligation time (naive, 2 days, 14 days, and 8 weeks post-SNL). Within theresulting 16 subgroups, both protein staining intensity measures andligation time factors represented naturally ordered values. Initial dataevaluation indicated the absence of normality in the distribution of thescored response, suggesting modeling by a nonparametric method. Thus aone-sided Jonckheere-Terpstra test was chosen to evaluate the basichypothesis that the distribution of mean protein expression scoreschanged across ligation time.

Results: Mechanical allodynia: SNL rats exhibited a dramatic ipsilateraldecrease in Von Frey response threshold to less than 2 g and then 1 g bythree and ten days post lesion, respectively. This enhanced level oftactile sensitivity in the ipsilateral paw was retained throughout theeight week test period. Contralateral paw response thresholds were ingeneral similar to naive or prelesion responses, i.e., 13-15 g; however,enhanced sensitivities (8-10 g) were seen at three days post-lesion,possibly reflecting a generalized post-surgical effect. Naive animalresponses to Von Frey stimulation were within the 13-15 g range over theentire eight-week period, bilaterally.

Intensity of β1A labeling in DRG. A graphic presentation of the meanstaining intensities in each group of DRGs studied is shown in FIG. 4.In both nociceptive and sensory neurons, β1A immunolabeling increasedwith post-surgical time, this increase being most prominent in theipsilateral neurons. Table 1 lists the p values resulting fromJonckheere-Terpstra analysis of the intensity versus time relationship.Intensity of β1 labeling in DRG. A graphic presentation of the meanlabeling intensity for each group of DRGs studied is presented in FIG.5. Although β1 immunolabeling tended to increase with post-surgicaltime, this trend was of greater significance in the L4 than the L5 DRG(Table 1).

Subcellular distribution of β1A and β1 immunoreactivity. In naive rats,the sodium channel β1A and β1 subunits exhibited identical subcellularlocalizations, virtually all of the staining being diffusely distributedthroughout the cell. Post-SNL, however, the subcellular distributions ofthe subunits diverged markedly. Whereas staining for the β1 subunitremained uniformly distributed within the DRG neurons in which it waslocated, β1A localization, most notably in the nociceptive neurons,assumed an altered pattern of distribution post SNL; staining for theβ1A subunit became discreetly localized in an area proximal to the cellmembrane. Whereas virtually 100% of β1A staining was diffuselydistributed in DRG cells from naive rats, only 23% was homogeneouslydistributed in nociceptive, ipsilateral, L5, DRG neurons (Table 2). Inthese cells, 51% of the staining for β1A was localized proximal to thecell membrane. Ipsilateral sensory DRG neurons, as well as bothnociceptive and sensory contralateral neurons, also exhibited alteredsubcellular β1A distributions. In these cells, 58-76% of β1A stainingremained diffuse, about 20% appeared punctate or Nissl-like, and theremainder was located adjacent to the cell membrane (Table 2). Thus, inaddition to exhibiting differences in the intensity of their labeling inDRG neurons post-SNL, sodium channel β1A and β1 subunits differedmarkedly in their post-SNL subcellular localizations.

EXAMPLE 13

Screening and Identification of Small Molecules that Interact Directlywith VGSC β1A Subunit

One of the regulatory functions of the β1A subunit is to increasefunctional channels by recruitment of α subunits to the cell surface.Disruption of the normal function of the β_(1A) subunit may result indown regulation of functional sodium channel in DRG after nerve injury.Small molecules that interact with the β1A subunit are identified byCETEK capillary zone electrophoresis technology. First, the targetprotein, VGSC β_(1A) subunit was over-expressed in bacteria. The cDNA ofthe β1A subunit was subcloned into pET29b bacteria expression vector(Novagen). The expression construct was then transformed into E. ColiBL21 (DE3) and the expression of the β1A induced by adding IPTG at roomtemperature for 4 hours. A single fresh colony from a plate wasinoculated into 50 ml LB medium containing 15 μg/ml of Kanamycin, andincubated with shaking at 37° C. until the OD₆₀₀ reached 0.6. Theexpression of β1A subunit fusion protein was induced by adding IPTG tofinal concentration to 1 mM and incubated at room temperature for 4hours. For SDS-PAGA analysis, 100 μl of cultures before and after IPTGinduction were aliquoted. The cells were spun down, resuspended in 20 mlof SDS loading buffer and subjected to 4-20% SDS-PAGE. Afterelectrophoresis, the gel was stained by SimpleBlue™ SafeStain(Invitrogen) and destained overnight with water. Lane 1: high molecularweight protein marker (Bio-Rad), lane 2, total cell lysate beforeinduction, and lane 3 total cell lysate after induction.

The resulting fusion protein of the β1A subunit had an S-tag, a 15 aminoacid motif, at its N-terminus and six histidines at its C-terminus. Thefusion protein is then affinity purified with a Ni⁺ column, andnon-covalently labeled with a fluorophore at the S-tag. The mobilityshifts of the β_(1A) subunit in capillary electrophoresis after bindingwith a small molecule that changes the conformation or surface charge ofthe target protein is detected with a laser-induced fluorescence.

EXAMPLE 14

Sodium Channel Functional Assay:

The regulatory activity of the β1A subunit is also studied by itsco-expression with the pore-forming and ion-conducting α subunit ineukaryotic cells, where sodium channel activity is tested by functionalbioassays. The α subunits are, but not limited to, Na_(V)1.2, Na_(V)1.3,Na_(V)1.6, Na_(V)1.8 and Na_(V)1.9, respectively. Determination ofsodium channel activity is performed by using two cell-based assays. Thefirst assay involves culturing cells in 96-well plates containing ascintillating base plate. The cell monolayers are stimulated withveratridine or any other appropriate sodium channel activator in thepresence of [¹⁴C]-guanidine. When the sodium channel is open, the[¹⁴C]-guanidinium ions flow down their concentration gradient into thecells. Since the cells are in close proximity to the scintillating base,light is emitted. The amount of light emitted is proportional to thelevel of sodium channel activity and is quantitated using ascintillation counter. The second assay utilizes a fluorescent,potential-sensitive dye. Monolayers of cells are loaded with thepotential-sensitive dye for 30 min at 37° C. After this incubationperiod, the cells are stimulated with veratridine or any other sodiumchannel activator, and the change in fluorescence is monitored by eithera fluorimeter, fluorescent imaging plate reader (FLIPR) or by afluorimeter-based cell imaging system. When the cells depolarize, thedye associates with cell membrane, resulting in increased fluorescence.When the cells hyperpolarize, the dye disassociates from the membrane,resulting in decreased fluorescence.

EXAMPLE 15

Cell Adhesion Assay:

Because the sodium channel β subunits have been implicated in homophiliccell-cell adhesion, the cell adhesion function of the β1A subunit istested. The adhesion assay utilizes L cell mouse fibroblasts, which lackmost of the macromolecules required for cell-cell adhesion. These Lcells are stably or transiently transfected with β1A and plated in96-well plates. Another set of L cell-β1A transfectants are loaded witha viable fluorescent dye and added to the cultures in the 96 well platesin the presence or absence of candidate compounds. After a determinedincubation period, the plates are placed in a fluorescence plate reader.If homophilic cell-cell adhesion has occurred, a detectable increase influorescence is observed. To determine specific activity, the signal iscompared to the fluorescence observed using untransfected L cellslabeled with viable fluorescent dye. TABLE 1 Statistical analyses oftrends over time post-SNL in the level of β1A and β1 expression in DRGneurons. The p values given were obtained with one-tailed,Jonckheere-Terpstra analyses of the data underlying FIGS. 9 and 10. SideRelative p value DRG to Ligation DRG Neuron Type β1A β1 L5 ipsilateralnociceptive 0.003* 0.058 sensory 0.011* 0.058 contralateral nociceptive0.029* 0.042* sensory 0.035* 0.097 L4 ipsilateral nociceptive 0.007*0.023* sensory 0.006* 0.001* contralateral nociceptive 0.001* 0.041*sensory 0.036* 0.019**p value < 0.05

TABLE 2 Subcellular distribution of β1A and β1 staining in L5 DRGneurons two weeks post-SNL. Side Relative Staining Percent of TotalAntibody to Ligation Distribution Nociceptive Sensory β1A IpsilateralMembrane 51 11 Punctate 26 18 Homogeneous 23 71 Contralateral Membrane28 4 Punctate 14 20 Homogeneous 58 76 β1 Ipsilateral Membrane 0 0Punctate 0 0 Homogeneous 100 100 Contralateral Membrane 0 0 Punctate 0 0Homogeneous 100 100

REFERENCES

-   Balser J R (1999) “Structure and function of the cardiac sodium    channels” Cardiovasc Res. 42: 327-38.-   Catterall W A (1992) Cellular and molecular biology of voltage-gated    sodium channels. Physiol Rev 72:S15-S48.-   Catterall W A (1993) Structure and function of voltage-gated ion    channels. Trends Neurosci 16:500-506.-   Chabal C, Russel L C, Burchiel K J (1989) The effect of intravenous    lidocaine, tocainide, and mexiletine on spontaneously active fibers    originating in rat sciatic neuromas. Pain 38:333-338.-   Chaplan S R, Bach F W, Pogrel J W, L L J M, Yaksh T L. Quantitative    assessment of tactile allodynia in the rat paw. J Neurosci Meth    1994; 53:55-63.-   D'Andrea M R, Derian C K, Leturcq D, Baker S M, Brunmark A, Ling P,    Darrow A L, Santulli R J, Brass L F, Andrade-Gordon P.    Characterization of protease activated receptor (PAR-2)    immunoreactivity in normal human tissues. J. Histochem. Cytochem.    1998; 46:157-164.-   Devor M, Keller C H, Deerinck T J, Ellisman M H. (1989) Na+ channel    accumulation on axolemma of afferent endings in nerve end neuromas    in Apteronotus. Neurosci Lett 102:149-154.-   Devor M, Wall P D, Catalan N (1992) Systematic lidocaine silences    ectopic neuroma and DRG discharge without blocking nerve conduction.    Pain 48:261-268.-   Devor M (1994) The pathophysiology of damaged peripheral nerves. In    Texbook of Pain, eds. Wall P D, Melzack R. (Churchill Liningstone,    Edinburgh), 2^(nd) Ed., pp. 79-101.-   Dib-Hajj S, Black J A, Felts P, Waxman S G (1996) Down-regulation of    transcripts for Na channel α-SNS in spinal sensory neurons following    axotomy. PNAS 93:14950-14954.-   England J D, Gamboni F, Ferguson M A, Levinson S R (1994) Sodium    channels accumulate at the tips of injured axons. Muscle Nerve    17:593-598.-   England J D, Happel L T, Kline D G, Gamboni F, Thouron C L, Liu Z P,    Levinson S R (1996) Sodium channel accumulation in humans with    painful neuromas. Neurology 4:272-276.-   Gould H J 3^(rd), England J D, Liu Z P, Levinson S R. Rapid sodium    channel augmentation in response to inflammation induced by complete    Freund's adjuvant. Brain Research 1998; 802:69-74.-   Isom L L, De Jongh K S, Patton D E, Reber B F X, Offord J,    Charbonneau H, Walsh K. Goldin A L and Catterall W A (1992) Primary    Structural and Functional Expression of the β1 Subunit of the Rat    Brain Sodium Channel” Science, 256; 839-842.-   Isom L L, De Jongh K S, Catterall W A (1994) Auxiliary β subunits of    voltage-gated ion channels. Neuron 12:1183-1194.-   Isom L L, Ragsdale D S, De Jongh K S, Westenbroek R E, Reber B F X,    Scheuer T, Catterall W A (1995) Structure and function of the β2    subunit of brain sodium channels, a transmembrane glycoprotein with    a CAM motif. Cell 83:433-442.-   Isom L L, Catterall W A (1996) “Na+ channel subunits and Ig domains”    Nature; 383(6598): 307-8.-   Kazen-Gillespie K, Ragsdale D S, D'Andrea M R, Laura N. Mattei,    Rogers K E, Isom L L (2000) Cloning, localization, and functional    expression of sodium channel β1α subunits. J Biol. Chem    275:2:1079-1088.-   Kazen-Gillespie K, Ragsdale D S, D'Andrea M R, Rogers K E, Isom L L.    Cloning, localization, and functional expression of sodium channel    β1A subunits. J Biol Chem 2000; 275:1-12.-   Kim S H, Chung J M (1992) An experimental model for peripheral    neuropathy produced by segmental spinal nerve ligation in the rat.    Pain 50:355-363.-   Kim S H, Chung J M. An experimental model for peripheral neuropathy    produced by segmental spinal nerve ligation in the rat. Pain 1992;    50:355-363.-   Marban E, Yamagishi T, Tomaselli G F J Physiol (Lond) (1998)    “Structure and function of voltage-gated sodium channels” 508 (Pt    3):647-57.-   Matzner O, Devor M (1992) Na⁺ conductance and the threshold for    repetitive neuronal firing. Brain Res 597:92-98.-   Matzner O, Devor M (1994) Hyperexcitability at sites of nerve injury    depends on voltage-sensitive Na+ channels. J Neurophysiol    72:349-359.-   Sutkowski E M and Catterall W A (1990) “β1 subunits of sodium    channels” J. Biol. Chem. 265; 12393-12399.-   Nordin M, Nystrom B, Wallin U, Hagbarth K-E (1984) Ectopic sensory    discharges and paresthesiae in patients with disorders of peripheral    nerves, dorsal roots and dorsal columns. Pain 20:231-245.-   Ochoa J, Torebjork H E (1980) Paresthesiae from ectopic impulse    generation in human sensory nerves. Brain 103:835-854.-   Oh Y, Sashihara S, Black J A, Waxman S G. Na⁺ channel β1 subunit    mRNA: differential expression in rat spinal sensory neurons. Mol    Brain Res 1995; 30:357-361.-   Omana-Zapata I, Khabbaz M A, Hunter J C, Clarke D E, Bley K R (1997)    Tetrodotoxin inhibits neuropathic ectopic activity in neuromas,    dorsal root ganglia and dorsal horn neurons. Pain 72(1-2):41-9.-   Porreca F, Lai J, Bian D, Wegert S, Ossipov M H, Eglen R M,    Kassotakis L, Novakovic S, Rabert D K, Sangameswaran L, Hunter J    C (1999) “A comparison of the potential role of the    tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in    rat models of chronic pain” Proc Natl Acad Sci USA; 96: 7640-4.-   Rizzo M A (1997) Successful treatment of painful mononeuropathy with    carbamazepine: insights into a possible molecular pain mechanism. J    Neurol Sci 152:103-106.-   Tanaka M, Cummins T R, Ishikawa K, Dib-Hajj S D, Black J A, Waxman S    G (1998) SNS Na+ channel expression increases in dorsal root    ganglion neurons in the carrageenan inflammatory pain model.    NeuroReport 9(6):967-72.-   Wallace R H, Wang D W, Singh R, Scheffer I E, George A L Jr,    Phillips H A, Saar K, Reis A, Johnson E W, Sutherland G R, Berkovic    S F, Mulley J C (1998) “Febrile seizures and generalized epilepsy    associated with a mutation in the Na+-channel beta1 subunit gene    SCN1B” Nature Genetics; 19: 366-70.-   Waxman S G, Brill M H (1978) Conduction through demylineated plaques    in multiple sclerosis: computer simulations of facilitation by short    internodes. J Neurol Neurosurg 41:408-417.-   Waxman S G, Kocsis J D, Black J A (1994) Type III sodium channel    mRNA is expressed in embryonic but not adult spinal sensory neurons,    and is reexpressed following axotomy. J Neurophysiol 72:466-470.-   Woolf C J, Safieh-Garabedian B, Ma Q-P, Crilly P, Winters J (1994)    Nerve growth factor contributes to the generation of inflammatory    sensory hypersensitivity. Neuroscience 62:327-331.-   Waxman S G, Dib-Hajj S, Cummins T R, Black J A. Sodium channels and    pain. PNAS 1999; 96(14):7635-9.

1-35. (canceled)
 36. An isolated protein encoded by a nucleic acidsequence capable of hybridizing under stringent hybridization conditionsto a nucleotide sequence having the sequence of SEQ ID NO:12 that whencombined with a Human α sodium channel subunit protein in a cell permitssodium ion flux in the cell.
 37. The protein according to claim 36,having an amino acid sequence selected from a group consisting of:(SEQ.ID.NO.:14) and functional derivatives thereof.
 38. A monospecificantibody immunologically reactive with a human β1A sodium channelsubunit protein.
 39. A compound that modulates the function of human β1Aselected by a method of screening for a modulator of sodium channelactivity comprising: (a) providing a cell that co-expresses a proteinencoded by SEQ ID NO:12, and a sodium channel α subunit protein, whereinthe cell elicits a sodium ion flux; (b) contacting the cell with aputative β1A modulating compound; (c) measuring a change upon the cellthat alters the sodium ion flux; and (d) comparing said change to a basevalue observed in an otherwise identical cell that does not express saidencoded protein.
 40. A pharmaceutical composition comprising a compoundof claim
 39. 41. A method of treating neuropathic pain in a patient inneed of such treatment comprising administration of a modulatingcompound of claim
 39. 42. A method of treating neuropathic pain in apatient in need of such treatment comprising altering the level of ahuman β1A subunit in a dorsal root ganglia cell in the patient.
 43. Amethod of treating chronic pain in a patient in need of such treatmentcomprising administering the compound of claim
 39. 44. A method oftreating febrile seizures in a patient in need of such treatmentcomprising administering the compound of claim
 39. 45. A method oftreating general epilepsy in a patient in need of such treatmentcomprising administering the compound of claim
 38. 46. An anticonvulsantpharmaceutical composition comprising a compound of claim
 39. 47. Amethod of treating arrhythmia in a patient in need of such treatmentcomprising administering the compound of claim
 38. 48. A pharmaceuticalcomposition comprising a compound that modulates the function of humanβ1A for use as a local anesthetic.
 49. A method for decreasingneuropathic pain in an individual comprising administering to saidindividual a modulator of a sodium channel β1A subunit in an amounteffective to change the sodium channel activity in said individual. 50.The method of claim 48 wherein said modulator decreases the expressionof sodium channel β1A subunit in the cells of said individual.
 51. Amethod for treating neuropathic pain in a subject comprising alteringthe level of sodium channel β1A subunits on the surface of a cell in asubject.
 52. A method for decreasing neuropathic pain in a humancomprising the step of administering a sodium channel β1Asubunit-binding molecule to a sodium channel β1A subunit-expressing cellin the human.